Nucleic acid and corresponding protein entitled 205P1B5 useful in treatment and detection of cancer

ABSTRACT

A novel gene (designated 205P1B5) and its encoded protein are described. While 205P1B5 exhibits tissue specific expression in normal adult tissue, it is aberrantly expressed in prostrate cancer. Consequently, 205P1B5 provides a diagnostic, prognostic, prophylactic and/or therapeutic target for cancer. The 205P1B5 gene or fragment thereof, or its encoded protein or a fragment thereof, can be used to elicit an immune response.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a national stage application under 35 USC §371 ofInternational Patent Application No. PCT/US02/27760, having anInternational Filing Date of Aug. 30, 2002 and published in the Englishlanguage on Mar. 13, 2003, which claims the priority benefit of U.S.Provisional Patent Application No. 60/316,664 filed Aug. 31, 2001, nowabandoned. The contents of those applications are hereby incorporated byreference herein in their entirety.

FIELD OF THE INVENTION

The invention described herein relates to a novel gene and its encodedprotein, termed 205P1B5, and to diagnostic and therapeutic, methods andcompositions useful in the management of various cancers that express205P1B5.

BACKGROUND OF THE INVENTION

Cancer is the second leading cause of human death next to coronarydisease. Worldwide, millions of people die from cancer every year. Inthe United States alone, as reported by the American Cancer Society,cancer causes the death of well over a half-million people annually,with over 1.2 million new cases diagnosed per year. While deaths fromheart disease have been declining significantly, those resulting fromcancer generally are on the rise. In the early part of the next century,cancer is predicted to become the leading cause of death.

Worldwide, several cancers stand out as the leading killers. Inparticular, carcinomas of the lung, prostate, breast, colon, pancreas,and ovary represent the primary causes of cancer death. These andvirtually all other carcinomas share a common lethal feature. With veryfew exceptions, metastatic disease from a carcinoma is fatal. Moreover,even for those cancer patients who initially survive their primarycancers, common experience has shown that their lives are dramaticallyaltered. Many cancer patients experience strong anxieties driven by theawareness of the potential for recurrence or treatment failure. Manycancer patients experience physical debilitations following treatment.Furthermore, many cancer patients experience a recurrence.

Worldwide, prostate cancer is the fourth most prevalent cancer in men.In North America and Northern Europe, it is by far the most commoncancer in males and is the second leading cause of cancer death in men.In the United States alone, well over 30,000 men die annually of thisdisease-second only to lung cancer. Despite the magnitude of thesefigures, there is still no effective treatment for metastatic prostatecancer. Surgical prostatectomy, radiation therapy, hormone ablationtherapy, surgical castration and chemotherapy continue to be the maintreatment modalities. Unfortunately, these treatments are ineffectivefor many and are often associated with undesirable consequences.

On the diagnostic front, the lack of a prostate tumor marker that canaccurately detect early-stage, localized tumors remains a significantlimitation in the diagnosis and management of this disease. Although theserum prostate specific antigen (PSA) assay has been a very useful tool,however its specificity and general utility is widely regarded aslacking in several important respects.

Progress in identifying additional specific markers for prostate cancerhas been improved by the generation of prostate cancer xenografts thatcan recapitulate different stages of the disease in mice. The LAPC (LosAngeles Prostate Cancer) xenografts are prostate cancer xenografts thathave survived passage in severe combined immune deficient (SCBD) miceand have exhibited the capacity to mimic the transition from androgendependence to androgen independence (Klein et al., 1997, Nat. Med.3:402). More recently identified prostate cancer markers include PCTA-1(Su et al., 1996, Proc. Natl. Acad. Sci. USA 93: 7252),prostate-specific membrane (PSM) antigen (Pinto et al., Clin Cancer Res1996 Sep. 2 (9): 1445-51), STEAP (Hubert, et al., Proc Natl Acad SciUSA. 1999 Dec. 7; 96(25): 14523-8) and prostate stem cell antigen (PSCA)(Reiter et al., 1998, Proc. Natl. Acad. Sci. USA 95: 1735).

While previously identified markers such as PSA, PSM, PCTA and PSCA havefacilitated efforts to diagnose and treat prostate cancer, there is needfor the identification of additional markers and therapeutic targets forprostate and related cancers in order to further improve diagnosis andtherapy.

Renal cell carcinoma (RCC) accounts for approximately 3 percent of adultmalignancies. Once adenomas reach a diameter of 2 to 3 cm, malignantpotential exists. In the adult, the two principal malignant renal tumorsare renal cell adenocarcinoma and transitional cell carcinoma of therenal pelvis or ureter. The incidence of renal cell adenocarcinoma isestimated at more than 29,000 cases in the United States, and more than11,600 patients died of this disease in 1998. Transitional cellcarcinoma is less frequent, with an incidence of approximately 500 casesper year in the United States.

Surgery has been the primary therapy for renal cell adenocarcinoma formany decades. Until recently, metastatic disease has been refractory toany systemic therapy. With recent developments in systemic therapies,particularly immunotherapies, metastatic renal cell carcinoma may beapproached aggressively in appropriate patients with a possibility ofdurable responses. Nevertheless, there is a remaining need for effectivetherapies for these patients.

Of all new cases of cancer in the United States, bladder cancerrepresents approximately 5 percent in men (fifth most common neoplasm)and 3 percent in women (eighth most common neoplasm). The incidence isincreasing slowly, concurrent with an increasing older population. In1998, there was an estimated 54,500 cases, including 39,500 in men and15,000 in women. The age-adjusted incidence in the United States is 32per 100,000 for men and 8 per 100,000 in women. The historic male/femaleratio of 3:1 may be decreasing related to smoking patterns in women.There were an estimated 11,000 deaths from bladder cancer in 1998 (7,800in men and 3,900 in women). Bladder cancer incidence and mortalitystrongly increase with age and will be an increasing problem as thepopulation becomes more elderly.

Most bladder cancers recur in the bladder. Bladder cancer is managedwith a combination of transurethral resection of the bladder (TUR) andin transvesical chemotherapy or immunotherapy. The multifocal andrecurrent nature of bladder cancer points out the limitations of TURMost muscle-invasive cancers are not cured by TUR alone. Radicalcystectomy and urinary diversion is the most effective means toeliminate the cancer but carry an undeniable impact on urinary andsexual function. There continues to be a significant need for treatmentmodalities that are beneficial for bladder cancer patients.

An estimated 130,200 cases of colorectal cancer occurred in 2000 in theUnited States, including 93,800 cases of colon cancer and 36,400 ofrectal cancer. Colorectal cancers are the third most common cancers inmen and women. Incidence rates declined significantly during 1992-1996(-2.1% per year). Research suggests that these declines have been due toincreased screening and polyp removal, preventing progression of polypsto invasive cancers. There were an estimated 56,300 deaths (47,700 fromcolon cancer, 8,600 from rectal cancer) in 2000, accounting for about11% of all U.S. cancer deaths.

At present, surgery is the most common form of therapy for colorectalcancer, and for cancers that have not spread, it is frequently curative.Chemotherapy, or chemotherapy plus radiation is given before or aftersurgery to most patients whose cancer has deeply perforated the bowelwall or has spread to the lymph nodes. A permanent colostomy (creationof an abdominal opening for elimination of body wastes) is occasionallyneeded for colon cancer and is infrequently required for rectal cancer.There continues to be a need for effective diagnostic and treatmentmodalities for colorectal cancer.

There were an estimated 164,100 new cases of lung and bronchial cancerin 2000, accounting for 14% of all U.S. cancer diagnoses. The incidencerate of lung and bronchial cancer is declining significantly in men,from a high of 86.5 per 100,000 in 1984 to 70.0 in 1996. In the 1990s,the rate of increase among women began to slow. In 1996, the incidencerate in women was 42.3 per 100,000.

Lung and bronchial cancer caused an estimated 156,900 deaths in 2000,accounting for 28% of all cancer deaths. During 1992-1996, mortalityfrom lung cancer declined significantly among men (−1.7% per year) whilerates for women were still significantly increasing (0.9% per year).Since 1987, more women have died each year of lung cancer than breastcancer, which, for over 40 years, was the major cause of cancer death inwomen. Decreasing lung cancer incidence and mortality rates most likelyresulted from decreased smoking rates over the previous 30 years;however, decreasing smoking patterns among women lag behind those ofmen. Of concern, although the declines in adult tobacco use have slowed,tobacco use in youth is increasing again.

Treatment options for lung and bronchial cancer are determined by thetype and stage of the cancer and include surgery, radiation therapy, andchemotherapy. For many localized cancers, surgery is usually thetreatment of choice. Because the disease has usually spread by the timeit is discovered, radiation therapy and chemotherapy are often needed incombination with surgery. Chemotherapy alone or combined with radiationis the treatment of choice for small cell lung cancer; on this regimen,a large percentage of patients experience remission, which in some casesis long lasting. There is however, an ongoing need for effectivetreatment and diagnostic approaches for lung and bronchial cancers.

An estimated 182,800 new invasive cases of breast cancer were expectedto occur among women in the United States during 2000. Additionally,about 1,400 new cases of breast cancer were expected to be diagnosed inmen in 2000. After increasing about 4% per year in the 1980s, breastcancer incidence rates in women have leveled off in the 1990s to about110.6 cases per 100,000.

In the U.S. alone, there were an estimated 41,200 deaths (40,800 women,400 men) in 2000 due to breast cancer. Breast cancer ranks second amongcancer deaths in women. According to the most recent data, mortalityrates declined significantly during 1992-1996 with the largest decreasesin younger women, both white and black. These decreases were probablythe result of earlier detection and improved treatment.

Taking into account the medical circumstances and the patient'spreferences, treatment of breast cancer may involve lumpectomy (localremoval of the tumor) and removal of the lymph nodes under the arm;mastectomy (surgical removal of the breast) and removal of the lymphnodes under the arm; radiation therapy, chemotherapy; or hormonetherapy. Often, two or more methods are used in combination. Numerousstudies have shown that, for early stage disease, long-term survivalrates after lumpectomy plus radiotherapy are similar to survival ratesafter modified radical mastectomy. Significant advances inreconstruction techniques provide several options for breastreconstruction after mastectomy. Recently, such reconstruction has beendone at the same time as the mastectomy.

Local excision of ductal carcinoma in situ (DCIS) with adequate amountsof surrounding normal breast tissue may prevent the local recurrence ofthe DCIS. Radiation to the breast and/or taroxifen may reduce the chanceof DCIS occurring in the remaining breast tissue. This is importantbecause DCIS, if left untreated, may develop into invasive breastcancer. Nevertheless, there are serious side effects or sequelae tothese treatments. There is, therefore, a need for efficacious breastcancer treatments.

There were an estimated 23,100 new cases of ovarian cancer in the UnitedStates in 2000. It accounts for 4% of all cancers among women and rankssecond among gynecologic cancers. During 1992-1996, ovarian cancerincidence rates were significantly declining. Consequent to ovariancancer, there were an estimated 14,000 deaths in 2000. Ovarian cancercauses more deaths than any other cancer of the female reproductivesystem.

Surgery, radiation therapy, and chemotherapy are treatment options forovarian cancer. Surgery usually includes the removal of one or bothovaries, the fallopian tubes (salpingo-oophorectomy), and the uterus(hysterectomy). In some very early tumors, only the involved ovary willbe removed, especially in young women who wish to have children. Inadvanced disease, an attempt is made to remove all intra-abdominaldisease to enhance the effect of chemotherapy. There continues to be animportant need for effective treatment options for ovarian cancer.

There were an estimated 28,300 new cases of pancreatic cancer in theUnited States in 2000. Over the past 20 years, rates of pancreaticcancer have declined in men. Rates among women have remainedapproximately constant but may be beginning to decline. Pancreaticcancer caused an estimated 28,200 deaths in 2000 in the United States.Over the past 20 years, there has been a slight but significant decreasein mortality rates among men (about −0.9% per year) while rates haveincreased slightly among women.

Surgery, radiation therapy, and chemotherapy are treatment options forpancreatic cancer. These treatment options can extend survival and/orrelieve symptoms in many patients but are not likely to produce a curefor most. There is a significant need for additional therapeutic anddiagnostic options for pancreatic cancer.

SUMMARY OF TIE INVENTION

The present invention relates to a gene, designated 205P1B5, that isover-expressed in the cancer(s) listed in Table I. There are twovariants of 205P1B5 (see, e.g. FIG. 2); unless the context clearlyindicates otherwise. Reference herein to 205P1B5 refers to either ofthese variants. Northern blot expression analysis of 205P1B5 geneexpression in normal tissues shows a restricted expression pattern inadult tissues. The nucleotide (FIG. 2) and amino acid (FIG. 2, and FIG.3) sequences of 205P1B5 are provided. The tissue-related profile of205P1B5 in normal adult tissues, combined with the over-expressionobserved in prostate tumors, shows that 205P1B5 is aberrantlyover-expressed in at least some cancers, and thus serves as a usefuldiagnostic, prophylactic, prognostic, and/or therapeutic target forcancers of the tissue(s) such as those listed in Table I.

The invention provides polynucleotides corresponding or complementary toall or part of the 205P1B5 genes, mRNAs, and/or coding sequences,preferably in isolated form, including polynucleotides encoding205P1B5-related proteins and fragments of 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or more tan 25contiguous amino acids; at least 30, 35, 40, 45, 50, 55, 60, 65, 70, 80,85, 90, 95, 100or more than 100 contiguous amino acids of a205P1B5-related protein, as well as the peptides/proteins themselves;DNA, RNA, DNA/RNA hybrids, and related molecules, polynucleotides oroligonucleotides complementary or having at least a 90% homology to the205P1B5 genes or mRNA sequences or parts thereof, and polynucleotides oroligonucleotides that hybridize to the 205P1B5 genes, mRNAs, or to205P1B5-encoding polynucleotides. Also provided are means for isolatingcDNAs and the genes encoding 205P1B5. Recombinant DNA moleculescontaining 205P1B5 polynucleotides, cells transformed or transduced withsuch molecules, and host-vector systems for the expression of 205P1B5gene products are also provided. The invention further providesantibodies that bind to 205P1B5 proteins and polypeptide fragmentsthereof, including polyclonal and monoclonal antibodies, murine andother mammalian antibodies, chimeric antibodies, humanized and fullyhuman antibodies, and antibodies labeled with a detectable marker. Incertain embodiments there is a proviso that the entire nucleic acidsequence of FIG. 2 is not encoded and/or the entire amino acid sequenceof FIG. 2 is not prepared. In certain embodiments, the entire nucleicacid sequence of FIG. 2 is encoded and/or the entire amino acid sequenceof FIG. 2 is prepared, either of which are in respective human unit doseforms.

The invention further provides methods for detecting the presence andstatus of 205P1B5 polynucleotides and proteins in various biologicalsamples, as well as methods for identifying cells that express 205P1B5.A typical embodiment of this invention provides methods for monitoring205P1B5 gene products in a tissue or hematology sample having orsuspected of having some form of growth dysregulation such as cancer.

The invention further provides various immunogenic or therapeuticcompositions and strategies for treating cancers that express 205P1B5such as prostate cancers, including therapies aimed at inhibiting thetranscription, translation, processing or function of 205P1B5 as well ascancer vaccines.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. 205P1B5 SSH sequence. The 205P1B5 SSH sequence (SEQ ID NO.:736).

FIG. 2. The cDNA (SEQ ID. NO.:701) and amino acid sequence (SEQ ID.NO.:702) of 205P1B5 v.1 and the cDNA (SEQ ID. NO.:703) and amino acidsequence (SEQ ID. NO.:704) of 205P1B5 v.2. The start methionine isunderlined. The open reading frame extends from nucleic acid 555 to 2144including the stop codon.

FIG. 3. Amino acid sequence of 205P1B5 v.1 (SEQ ID. NO.:702) and aminoacid sequence of 205P1B5 v.2 (SEQ ID. NO.:704). Each 205P1B5 protein has529 amino acids.

FIG. 4. Sequence alignment of 205P1B5v.1 (SEQ ID NO.:702) with GenBankaccession number (SEQ ID. NO.:705).

FIG. 5. Hydrophilicity amino acid profile of 205P1B5 determined bycomputer algorithm sequence analysis using the method of Hopp and Woods(Hopp T. P., Woods K. R., 1981. Proc. Natl. Acad. Sci. U.S.A.78:3824-3828) accessed on the Protscale website (world wide web URLch/cgi-bin/protscale.pl) through the ExPasy molecular biology server.

FIG. 6. Hydropathicity amino acid profile of 205P1B5 determined bycomputer algorithm sequence analysis using the method of Kyte andDoolittle (Kyte J., Doolittle R. F., 1982. J. Mol. Biol. 157:105-132)accessed on the ProtScale website (world wide web URLexpasy.ch/cgi-bin/protscale.pl) through the ExPasy molecular biologyserver.

FIG. 7. Percent accessible residues amino acid profile of 205P1B5determined by computer algorithm sequence analysis using the method ofJanin (Janin J., 1979 Nature 277:491-492) accessed on the ProtScalewebsite (world wide web URL expasy.ch/cgi-bin/protscale.pl) through theExPasy molecular biology server.

FIG. 8. Average flexibility amino acid profile of 205P1B5 determined bycomputer algorithm sequence analysis using the method of Bhaskaran andPonnuswamy (Bhaskaran R, and Ponnuswamy P. K., 1988. Int. J. Pept.Protein Res. 32:242-255) accessed on the ProtScale website (world wideweb URL expasy.ch/cgi-bin/protscale.pl) through the ExPasy molecularbiology server.

FIG. 9. Beta-turn amino acid profile of 205P1B5 determined by computeralgorithm sequence analysis using the method of Deleage and Roux(Deleage, G., Roux B. 1987 Protein Engineering 1:289-294) accessed onthe ProtScale website (world wide web URLexpasy.ch/cgi-bin/protscale.pl) through the ExPasy molecular biologyserver.

FIG. 10. RT-PCR analysis of 205P1B5 expression. First strand cDNA wasprepared from vital pool 1 (VP: live lung and kidney), vital pool 2(VP2: pancreas, colon and stomach), prostate xenograft pool (LAPC-4AD,LAPC-4AI, LAPC-9AD, LAPC-9AI), prostate cancer pool, and cancermetastasis pool. Normalization was performed by PCR using primers toactin and GAPDH. Semi-quantitative PCR, using primers to 205P1B5, wasperformed at 26 and 30 cycles of amplification. Results show expressionof 205P1B5 in prostate cancer pool, prostate xenograft pool, cancermetastasis pool, but not in VP1 and VP2.

FIG. 11. Expression of 205P1B5 in normal human tissues. Two multipletissue northern blots (Clontech) with 2 mg of mRNA/lane, were probedwith 205P1B5 sequences. Size standards in kilobases (kb) are indicatedon the side. The results show restricted expression of an approximately5 kb 205P1B5 transcript (indicated with an arrow) in prostate and tolower level in brain tissues. A larger transcript of approximately 7.5kb in size is detected in liver.

FIG. 12. Expression of 205P1B5 in human patient cancer specimens. RNAwas extracted from a pool of 3 prostate cancer tumors, as well as fromnormal prostate (NP), normal bladder (NB), normal kidney (NK) and normalcolon (NC). Northern blots with 10 mg of total RNA/lane were probed with205P1B5 sequences. Size standards in kilobases (kb) are indicated on theside. The results show expression of 205P1B5 in prostate cancer pool andnormal prostate, but not in the other normal tissues.

FIG. 13. Expression of 205P1B5 in Prostate Cancer Xenografts andProstate Cancer Patient Specimens. RNA was extracted from prostatecancer xenografts (LAPC-4AD, LAPC-4AI, LAPC-9AD, LAPC-9AI), prostatecancer cell line PC3, normal prostate (N), prostate tumors (T) andnormal adjacent tissue (Nat) derived from prostate cancer patients.Northern blot with 10 mg of total RNA/lane was probed with the 205P1B5SSH sequence. Size standards in kilobases (kb) are indicated on theside. Results show expression of 205P1B5 in all prostate tumor specimenstested. Expression is also seen in 3 of the 4 xenografts, but not in thePC3 cell line.

FIG. 14. Secondary structure and transmembrane prediction for 205P1B5.Panel A. The secondary structure of 205P1B5v.1 protein (SEQ ID NO.:702)was depicted using the HNN—Hierarchical Neural Network method (Guermeur,1997, world wide web URLpbil.ibcp.fr/cgi-bin/npsa_automat.pl?page=npsa_nn.html), accessed fromthe ExPasy molecular biology server (world wide web URLexpasy.ch/tools/). This method indicates the presence and location ofalpha helices, extended strands, and random coils from the primaryprotein sequence. The percent of the protein in a given secondarystructure is also given. Panel B. Schematic representation oftransmembrane regions and orientation of 205P1B5 based on the TMpredalgorithm of Hofmann and Stoffel which utilizes TMBASE (K. Hofmann, W.Stoffel. TMBASE—A database of membrane spanning protein segments Biol.Chem. Hoppe-Seyler 374:166, 1993). Panel C. Schematic representation oftransmembrane regions and the extracellular and intracellularorientation of 205P1B5 based on the algorithm of Sonnhammer, von Heijne,and Krogh (Erik L. L., et al., A hidden Markov model for predictingtransmembrane helices in protein sequences. In Proc. of Sixth Int. Conf.on Intelligent Systems for Molecular Biology, p 175-182 Ed J. Glasgow,et al., Menlo Park, Calif.: AAAI Press, 1998). Both transmembraneprograms presented in Panel B and Panel C indicate that 205P1B5 contains5 transmembrane domains consistent with it being a G-protein coupledreceptor.

FIG. 15. Expression of 205P1B5 in cancer metastasis patient specimens.RNA was extracted from prostate cancer metastasis to lymph node obtainedfrom two different patients, as well as from normal bladder (NB), normalkidney (NK), normal lung (NL), normal breast (NBr), normal ovary (NO),and normal pancreas (NPa). Northern blots with 10 μg of total RNA/lanewere probed with 205P1B5 sequence. Size standards in kilobases (kb) areindicated on the side. The results show expression of 205P1B5 in bothcancer metastasis samples but not in the normal tissues tested.

FIG. 16. Enhanced Proliferation of Recombinant 3T3-205P1B5 Cells. Forthis data, control 3T3 and 3T3-205P1B5 cells were grown in 96 well platein 0.5 or 10% FBS. Proliferation was measured by Alamar blue after 48and 72 hours. Enhanced proliferation of 3T3-205P1B5 relative to controlcells is observed as early as 48 hours.

DETAILED DESCRIPTION OF THE INVENTION

Outline of Sections

I.) Definitions

II.) 205P1B5 Polynucleotides

-   -   II.A.) Uses of 205P1B5 Polynucleotides        -   II.A.1.) Monitoring of Genetic Abnormalities        -   II.A.2.) Antisense Embodiments        -   II.A.3.) Primers and Primer Pairs        -   II.A.4.) Isolation of 205P1B5-Encoding Nucleic Acid            Molecules        -   II.A.5.) Recombinant Nucleic Acid Molecules and Host-Vector            Systems

III.) 205P1B5-related Proteins

-   -   III.A.) Motif-bearing Protein Embodiments    -   III.B.) Expression of 205P1B5-related Proteins    -   III.C.) Modifications of 205P1B5-related Proteins    -   III.D.) Uses of 205P1B5-related Proteins

IV.) 205P1B5 Antibodies

V.) 205P1B5 Cellular Immune Responses

VI.) 205P1B5 Transgenic Animals

VII.) Methods for the Detection of 205P1B5

VIII.) Methods for Monitoring the Status of 205P1B5-related Genes andTheir Products

IX.) Identification of Molecules That Interact With 205P1B5

X.) Therapeutic Methods and Compositions

-   -   X.A.) Anti-Cancer Vaccines    -   X.B.) 205P1B5 as a Target for Antibody-Based Therapy    -   X.C.) 205P1B5 as a Target for Cellular Immune Responses        -   X.C.1. Minigene Vaccines        -   X.C.2. Combinations of CTL Peptides with Helper Peptides        -   X.C.3. Combinations of CTL Peptides with T Cell Priming            Agents        -   X.C.4. Vaccine Compositions Comprising DC Pulsed with CTL            and/or HTL Peptides    -   X.D.) Adoptive Immunotherapy    -   X.E.) Administration of Vaccines for Therapeutic or Prophylactic        Purposes

XI.) Diagnostic and Prognostic Embodiments of 205P1B5.

XII.) Inhibition of 205P1B5 Protein Function

-   -   XII.A.) Inhibition of 205P1B5 With Intracellular Antibodies    -   XII.B.) Inhibition of 205P1B5 with Recombinant Proteins    -   XII.C.) Inhibition of 205P1B5 Transcription or Translation    -   XII.D.) General Considerations for Therapeutic Strategies

XIII.) KITS

I.) Definitions:

Unless otherwise defined, all terms of art, notations and otherscientific terms or terminology used herein are intended to have themeanings commonly understood by those of skill in the art to which thisinvention pertains. In some cases, terms with commonly understoodmeanings are defined herein for clarity and/or for ready reference, andthe inclusion of such definitions herein should not necessarily beconstrued to represent a substantial difference over what is generallyunderstood in the art. Many of the techniques and procedures describedor referenced herein are well understood and commonly employed usingconventional methodology by those skilled in the art, such as, forexample, the widely utilized molecular cloning methodologies describedin Sambrook et al., Molecular Cloning: A Laboratory Manual 2nd. edition(1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. Asappropriate, procedures involving the use of commercially available kitsand reagents are generally carried out in accordance with manufacturerdefined protocols and/or parameters unless otherwise noted.

The terms “advanced prostate cancer”, “locally advanced prostatecancer”, “advanced disease” and “locally advanced disease” mean prostatecancers that have extended through the prostate capsule, and are meantto include stage C disease under the American Urological Association(AUA) system, stage C1-C2 disease under the Whitmore-Jewett system, andstage T3-T4 and N+ disease under the TNM (tumor, node, metastasis)system. In general, surgery is not recommended for patients with locallyadvanced disease, and these patients have substantially less favorableoutcomes compared to patients having clinically localized(organ-confined) prostate cancer. Locally advanced disease is clinicallyidentified by palpable evidence of induration beyond the lateral borderof the prostate, or asymmetry or induration above the prostate base.Locally advanced prostate cancer is presently diagnosed pathologicallyfollowing radical prostatectomy if the tumor invades or penetrates theprostatic capsule, extends into the surgical margin, or invades theseminal vesicles.

“Altering the native glycosylation pattern” is intended for purposesherein to mean deleting one or more carbohydrate moieties found innative sequence 205P1B5 (either by removing the underlying glycosylationsite or by deleting the glycosylation by chemical and/or enzymaticmeans), and/or adding one or more glycosylation sites that are notpresent in the native sequence 205P1B5. In addition, the phrase includesqualitative changes in the glycosylation of the native proteins,involving a change in the nature and proportions of the variouscarbohydrate moieties present.

The term “analog” refers to a molecule which is structurally similar orshares similar or corresponding attributes with another molecule (e.g. a205P1B5-related protein). For example an analog of the 205P1B5 proteincan be specifically bound by an antibody or T cell that specificallybinds to 205P1B5.

The term “antibody” is used in the broadest sense. Therefore an“antibody” can be naturally occurring or man-made such as monoclonalantibodies produced by conventional hybridoma technology. Anti-205P1B5antibodies comprise monoclonal and polyclonal antibodies as well asfragments containing the antigen-binding domain and/or one or morecomplementarity determining regions of these antibodies.

An “antibody fragment” is defined as at least a portion of the variableregion of the immunoglobulin molecule that binds to its target, i.e.,the antigen-binding region. In one embodiment it specifically coverssingle anti-205P1B5 antibodies and clones thereof (including agonist,antagonist and neutralizing antibodies) and anti-205P1B5 antibodycompositions with polyepitopic specificity.

The term “codon optimized sequences” refers to nucleotide sequences thathave been optimized for a particular host species by replacing anycodons having a usage frequency of less than about 20%. Nucleotidesequences that have been optimized for expression in a given hostspecies by elimination of spurious polyadenylation sequences,elimination of exon/intron splicing signals, elimination oftransposon-like repeats and/or optimization of GC content in addition tocodon optimization are referred to herein as an “expression enhancedsequences.”

The term “cytotoxic agent” refers to a substance that inhibits orprevents the function of cells and/or causes destruction of cells. Theterm is intended to include radioactive isotopes chemotherapeuticagents, and toxins such as small molecule toxins or enzymatically activetoxins of bacterial, fungal, plant or animal origin, including fragmentsand/or variants thereof. Examples of cytotoxic agents include, but arenot limited to maytansinoids, yttrium, bismuth, ricin, ricin A-chain,doxorubicin, daunorubicin, taxol, ethidium bromide, mitomycin,etoposide, tenoposide, vincristine, vinblastine, colchicine, dihydroxyanthracin dione, actinomycin, diphtheria toxin, Pseudomonas exotoxin(PE) A, PE40, abrin, abrin A chain, modeccin A chain, alpha-sarcin,gelonin, mitogellin, retstrictocin, phenomycin, enomycin, curicin,crotin, calicheamicin, sapaonaria officinalis inhibitor, andglucocorticoid and other chemotherapeutic agents, as well asradioisotopes such as At²¹¹, I¹³¹, I¹²⁵, Y⁹⁰, Re¹⁸⁶, Re¹⁸⁸, SM¹⁵³,Bi²¹², P³² and radioactive isotopes of Lu. Antibodies may also beconjugated to an anti-cancer pro-drug activating enzyme capable ofconverting the pro-drug to its active form.

The term “homolog” refers to a molecule which exhibits homology toanother molecule, by for example, having sequences of chemical residuesthat are the same or similar at corresponding positions.

“Human Leukocyte Antigen” or “HLA” is a human class I or class II MajorHistocompatibility Complex (MHC) protein (see, e.g., Stites, et al.,IMMUNOLOGY, 8^(Th) ED., Lange Publishing, Los Altos, Calif. (1994).

The terms “hybridize”, “hybridizing”, “hybridizes” and the like, used inthe context of polynucleotides, are meant to refer to conventionalhybridization conditions, preferably such as hybridization in 50%formamide/6×SSC/0.1% SDS/100 μg/ml mDNA, in which temperatures forhybridization are above 37 degrees C. and temperatures for washing in0.1×SSC/0.1% SDS are above 55 degrees C.

The phrases “isolated” or “biologically pure” refer to material which issubstantially or essentially free from components which normallyaccompany the material as it is found in its native state. Thus,isolated peptides in accordance with the invention preferably do notcontain materials normally associated with the peptides in their in situenvironment. For example, a polynucleotide is said to be “isolated” whenit is substantially separated from contaminant polynucleotides thatcorrespond or are complementary to genes other than the 205P1B5 gene orthat encode polypeptides other than 205P1B5 gene product or fragmentsthereof. A skilled artisan can readily employ nucleic acid isolationprocedures to obtain an isolated 205P1B5 polynucleotide. A protein issaid to be “isolated,” for example, when physical, mechanical orchemical methods are employed to remove the 205P1B5 protein fromcellular constituents that are normally associated with the protein. Askilled artisan can readily employ standard purification methods toobtain an isolated 205P1B5 protein. Alternatively, an isolated proteincan be prepared by chemical means.

The term “mammal” refers to any organism classified as a mammal,including mice, rats, rabbits, dogs, cats, cows, horses and humans. Inone embodiment of the invention, the mammal is a mouse. In anotherembodiment of the invention, the mammal is a human.

The terms “metastatic prostate cancer” and “metastatic disease” meanprostate cancers that have spread to regional lymph nodes or to distantsites, and are meant to include stage D disease under the AUA system andstage T×N×M+ under the TNM system. As is the case with locally advancedprostate cancer, surgery is generally not indicated for patients withmetastatic disease, and hormonal (androgen ablation) therapy is apreferred treatment modality. Patients with metastatic prostate cancereventually develop an androgen-refractory state within 12 to 18 monthsof treatment initiation. Approximately half of these androgen-refractorypatients die within 6 months after developing that status. The mostcommon site for prostate cancer metastasis is bone. Prostate cancer bonemetastases are often osteoblastic rather than osteolytic (i.e.,resulting in net bone formation). Bone metastases are found mostfrequently in the spine, followed by the femur, pelvis, rib cage, skulland humerus. Other common sites for metastasis include lymph nodes,lung, liver and brain. Metastatic prostate cancer is typically diagnosedby open or laparoscopic pelvic lymphadenectomy, whole body radionuclidescans, skeletal radiography, and/or bone lesion biopsy.

The term “monoclonal antibody” refers to an antibody obtained from apopulation of substantially homogeneous antibodies, i.e., the antibodiescomprising the population are identical except for possible naturallyoccurring mutations that are present in minor amounts.

A “motif”, as in biological motif of an 205P1B5-related protein, refersto any pattern of amino acids forming part of the primary sequence of aprotein, that is associated with a particular function (e.g.protein-protein interaction, protein-DNA interaction, etc.) ormodification (e.g. that is phosphorylated, glycosylated or amidated), orlocalization (e.g. secretory sequence, nuclear localization sequence,etc.) or a sequence that is correlated with being immunogenic, eitherhumoraly or cellularly. A motif can be either contiguous or capable ofbeing aligned to certain positions that are generally correlated with acertain function or property. In the context of HIA motifs, “motif”refers to the pattern of residues in a peptide of defined length,usually a peptide of from about 8 to about 13 amino acids for a class IHLA motif and from about 6 to about 25 amino acids for a class II HLAmotif, which is recognized by a particular HLA molecule. Peptide motifsfor HLA binding are typically different for each protein encoded by eachhuman HLA allele and differ in the pattern of the primary and secondaryanchor residues.

A “pharmaceutical excipient” comprises a material such as an adjuvant, acarrier, pH-adjusting and buffering agents, tonicity adjusting agents,wetting agents, preservative, and the like.

“Pharmaceutically acceptable” refers to a non-toxic, inert, and/orcomposition that is physiologically compatible with humans or othermammals.

The term “polynucleotide” means a polymeric form of nucleotides of atleast 10 bases or base pairs in length, either ribonucleotides ordeoxynucleotides or a modified form of either type of nucleotide, and ismeant to include single and double stranded forms of DNA and/or RNA. Inthe art, this term if often used interchangeably with “oligonucleotide”.A polynucleotide can comprise a nucleotide sequence disclosed hereinwherein thymidine (T) (as shown for example in SEQ ID NO: 702) can alsobe uracil (U); this definition pertains to the differences between thechemical structures of DNA and RNA, in particular the observation thatone of the four major bases in RNA is uracil (U) instead of thymidine(T).

The term “polypeptide” means a polymer of at least about 4, 5, 6, 7, or8 amino acids. Throughout the specification, standard three letter orsingle letter designations for amino acids are used. In the art, thisterm is often used interchangeably with “peptide” or “protein”.

An HLA “primary anchor residue” is an amino acid at a specific positionalong a peptide sequence which is understood to provide a contact pointbetween the immunogenic peptide and the HLA molecule. One to three,usually two, primary anchor residues within a peptide of defined lengthgenerally defines a “motif” for an immunogenic peptide. These residuesare understood to fit in close contact with peptide binding groove of anHLA molecule, with their side chains buried in specific pockets of thebinding groove. In one embodiment, for example, the primary anchorresidues for an HLA class I molecule are located at position 2 (from theamino terminal position) and at the carboxyl terminal position of a 8,9, 10, 11, or 12 residue peptide epitope in accordance with theinvention. In another embodiment for example, the primary anchorresidues of a peptide that will bind an HLA class II molecule are spacedrelative to each other, rather than to the termini of a peptide, wherethe peptide is generally of at least 9 amino acids in length. Theprimary anchor positions for each motif and supermotif are set forth inTable IV. For example, analog peptides can be created by altering thepresence or absence of particular residues in the primary and/orsecondary anchor positions shown in Table IV. Such analogs are used tomodulate the binding affinity and/or population coverage of a peptidecomprising a particular HLA motif or supermotif.

A “recombinant” DNA or RNA molecule is a DNA or RNA molecule that hasbeen subjected to molecular manipulation in vitro.

“Stringency” of hybridization reactions is readily determinable by oneof ordinary skill in the art and generally is an empirical calculationdependent upon probe length, washing temperature, and saltconcentration. In general longer probes require higher temperatures forproper annealing, while shorter probes need lower temperatures.Hybridization generally depends on the ability of denatured nucleic acidsequences to reanneal when complementary strands are present in anenvironment below their melting temperature. The higher the degree ofdesired homology between the probe and hybridizable sequence, the higherthe relative temperature that can be used. As a result, it follows thathigher relative temperatures would tend to make the reaction conditionsmore stringent, while lower temperatures less so. For additional detailsand explanation of stringency of hybridization reactions, see Ausubel etal., Current Protocols in Molecular Biology, Wiley IntersciencePublishers, (1995).

“Stringent conditions” or “high stringency conditions”, as definedherein, are identified by, but not limited to, those that (1) employ lowionic strength and high temperature for washing, for example 0.015 Msodium chloride/0.0015 M sodium citrate/0.1% sodium dodecyl sulfate at50° C.; (2) employ during hybridization a denaturing agent, such asformamide, for example, 50% (v/v) formamide with 0.1% bovine serumalbumin/0.1% Ficoll/0.1% polyvinylpyrrolidone/50 mM sodium phosphatebuffer at pH 6.5 with 750 mM sodium chloride, 75 mM sodium citrate at42° C.; or (3) employ 50% formamide, 5×SSC (0.75 M NaCl, 0.075 M sodiumcitrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate,5×Denhardt's solution, sonicated salmon sperm DNA (50 μg/ml), 0.1% SDS,and 10% dextran sulfate at 42° C., with washes at 42° C. in 0.2×SSC(sodium chloride/sodium citrate) and 50% formamide at 55° C., followedby a high-stringency wash consisting of 0.1×SSC containing EDTA at 55°C. “Moderately stringent conditions” are described by, but not limitedto, those in Sambrook et al., Molecular Cloning: A Laboratory Manual,New York: Cold Spring Harbor Press, 1989, and include the use of washingsolution and hybridization conditions (e.g., temperature, ionic strengthand % SDS) less stringent than those described above. An example ofmoderately stringent conditions is overnight incubation at 37° C. in asolution comprising: 20% formamide, 5×SSC (150 mM NaCl, 15 mM trisodiumcitrate), 50 mM sodium phosphate (pH 7.6), 5×Denhardt's solution, 10%dextran sulfate, and 20 mg/mL denatured sheared salmon sperm DNA,followed by washing the filters in 1×SSC at about 37-50° C. The skilledartisan will recognize how to adjust the temperature, ionic strength,etc. as necessary to accommodate factors such as probe length and thelike.

An HLA “supermotif” is a peptide binding specificity shared by HLAmolecules encoded by two or more HLA alleles.

As used herein “to treat” or “therapeutic” and grammatically relatedterms, refer to any improvement of any consequence of disease, such asprolonged survival, less morbidity, and/or a lessening of side effectswhich are the byproducts of an alternative therapeutic modality; fulleradication of disease is not required.

A “transgenic animal” (e.g., a mouse or rat) is an animal having cellsthat contain a transgene, which transgene was introduced into the animalor an ancestor of the animal at a prenatal, e.g., an embryonic stage. A“transgene” is a DNA that is integrated into the genome of a cell fromwhich a transgenic animal develops.

As used herein, an HLA or cellular immune response “vaccine” is acomposition that contains or encodes one or more peptides of theinvention. There are numerous embodiments of such vaccines, such as acocktail of one or more individual peptides; one or more peptides of theinvention comprised by a polyepitopic peptide; or nucleic acids thatencode such individual peptides or polypeptides, e.g., a minigene thatencodes a polyepitopic peptide. The “one or more peptides” can includeany whole unit integer from 1-150 or more, e.g., at least 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43,44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100,105, 110, 115, 120, 125, 130, 135, 140, 145, or 150 or more peptides ofthe invention. The peptides or polypeptides can optionally be modified,such as by lipidation, addition of targeting or other sequences. HLAclass I peptides of the invention can be admixed with, or linked to, HLAclass II peptides, to facilitate activation of both cytotoxic Tlymphocytes and helper T lymphocytes. HLA vaccines can also comprisepeptide-pulsed antigen presenting cells, e.g., dendritic cells.

The term “variant” refers to a molecule that exhibits a variation from adescribed type or norm, such as a protein that has one or more differentamino acid residues in the corresponding position(s) of a specificallydescribed protein (e.g. the 205P1B5 protein shown in FIG. 2 or FIG. 3).An analog is an example of a variant protein. Splice isoforms and singlenucleotides polymorphisms (SNPs) are further examples of variants.

The 205P1B5-related proteins of the invention include those specificallyidentified herein, as well as allelic variants, conservativesubstitution variants, analogs and homologs that can beisolated/generated and characterized without undue experimentationfollowing the methods outlined herein or readily available in the art.Fusion proteins that combine parts of different 205P1B5 proteins orfragments thereof, as well as fusion proteins of a 205P1B5 protein and aheterologous polypeptide are also included. Such 205P1B5 proteins arecollectively referred to as the 205P1B5-related proteins, the proteinsof the invention, or 205P1B5. The term “205P1B5-related protein” refersto a polypeptide fragment or an 205P1B5 protein sequence of 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, ormore than 25 amino acids; or, at least 30, 35, 40, 45, 50, 55, 60, 65,70, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145,150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 225, 250, 275,300, 325, 350, 375, 400, 425, 450, 475, 500, 525, or 529 or more aminoacids.

II.) 205P1B5 Polynucleotides

One aspect of the invention provides polynucleotides corresponding orcomplementary to all or part of an 205P1B5 gene, mRNA, and/or codingsequence, preferably in isolated form, including polynucleotidesencoding an 205P1B5-related protein and fragments thereof DNA, RNA,DNA/RNA hybrid, and related molecules, polynucleotides oroligonucleotides complementary to an 205P1B5 gene or mRNA sequence or apart thereof, and polynucleotides or oligonucleotides that hybridize toan 205P1B5 gene, mRNA, or to an 205P1B5 encoding polynucleotide(collectively, “205P1B5 polynucleotides”). In all instances whenreferred to in this section, T can also be U in FIG. 2.

Embodiments of a 205P1B5 polynucleotide include: a 205P1B5polynucleotide having the sequence shown in FIG. 2, the nucleotidesequence of 205P1B5 as shown in FIG. 2, wherein T is U; at least 10contiguous nucleotides of a polynucleotide having the sequence as shownin FIG. 2; or, at least 10 contiguous nucleotides of a polynucleotidehaving the sequence as shown in FIG. 2 where T is U. For example,embodiments of 205P1B5 nucleotides comprise, without limitation:

-   -   (a) a polynucleotide comprising or consisting of the sequence as        shown in FIG. 2 (SEQ ID NO.:701), wherein T can also be U;    -   (b) a polynucleotide comprising or consisting of the sequence as        shown in FIG. 2 (SEQ ID NO.:701), from nucleotide residue member        555 through nucleotide residue number 2144, wherein T can also        be U;    -   (c) a polynucleotide that encodes a 205P1B5-related protein        whose sequence is encoded by the cDNAs contained in the plasmid        designated ______ deposited with American Type Culture        Collection as Accession No. ______;    -   (d) a polynucleotide that encodes an 205P1B5-related protein        that is at least 90% homologous to the entire amino acid        sequence shown in FIG. 2 (SEQ ID NO.:702);    -   (e) a polynucleotide that encodes an 205P1B5-related protein        that is at least 90% identical to the entire amino acid sequence        shown in FIG. 2 (SEQ ID NO:702).    -   (f) a polynucleotide that encodes at least one peptide set forth        in Tables V-XVIII;    -   (g) a polynucleotide that encodes a peptide region of at least 5        amino acids of FIG. 3 in any whole number increment up to 529        that includes an amino acid position having a value greater than        0.5 in the Hydrophilicity profile of FIG. 5;    -   (h) a polynucleotide that encodes a peptide region of at least 5        amino acids of FIG. 3 in any whole number increment up to 529        that includes an amino acid position having a value less than        0.5 in the Hydropathicity profile of FIG. 6;    -   (i) a polynucleotide that encodes a peptide region of at least 5        amino acids of FIG. 3 in any whole number increment up to 529        that includes an amino acid position having a value greater than        0.5 in the Percent Accessible Residues profile of FIG. 7;    -   (j) a polynucleotide that encodes a peptide region of at least 5        amino acids of FIG. 3 in any whole number increment up to 529        that includes an amino acid position having a value greater than        0.5 in the Average Flexibility profile on FIG. 8;    -   (k) a polynucleotide that encodes a peptide region of at least 5        amino acids of FIG. 3 in any whole number increment up to 529        that includes an amino acid position having a value greater than        0.5 in the Beta-turn profile of FIG. 9;    -   (l) a polynucleotide that is fully complementary to a        polynucleotide of any one of (a)-(k);    -   (m) a polynucleotide that selectively hybridizes under stringent        conditions to a polynucleotide of (a)-(l);    -   (n) a peptide that is encoded by any of (a)-(k); and,    -   (o) a polynucleotide of any of (a)-(m)or peptide of (n) together        with a pharmaceutical excipient and/or in a human unit dose        form.

As used herein, a range is understood to specifically disclose all wholeunit positions thereof.

Typical embodiments of the invention disclosed herein include 205P1B5polynucleotides that encode specific portions of the 205P1B5 mRNAsequence (and those which are complementary to such sequences) such asthose that encode the protein and fragments thereof, for example of 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100,105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170,175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 250, 275, 300,325, 350, 375, 400, 425, 450, 475, 500, 525 or 529 contiguous aminoacids.

For example, representative embodiments of the invention disclosedherein include: polynucleotides and their encoded peptides themselvesencoding about amino acid 1 to about amino acid 10 of the 205P1B5protein shown in FIG. 2 or FIG. 3, polynucleotides encoding about aminoacid 10 to about amino acid 20 of the 205P1B5 protein shown in FIG. 2,or FIG. 3, polynucleotides encoding about amino acid 20 to about aminoacid 30 of the 205P1B5 protein shown in FIG. 2 or FIG. 3,polynucleotides encoding about amino acid 30 to about amino acid 40 ofthe 205P1B5 protein shown in FIG. 2 or FIG. 3, polynucleotides encodingabout amino acid 40 to about amino acid 50 of the 205P1B5 protein shownin FIG. 2 or FIG. 3, polynucleotides encoding about amino acid 50 toabout amino acid 60 of the 205P1B5 protein shown in FIG. 2 or FIG. 3,polynucleotides encoding about amino acid 60 to about amino acid 70 ofthe 205P1B5 protein shown in FIG. 2 or FIG. 3, polynucleotides encodingabout amino acid 70 to about amino acid 80 of the 205P1B5 protein shownin FIG. 2 or FIG. 3, polynucleotides encoding about amino acid 80 toabout amino acid 90 of the 205P1B5 protein shown in FIG. 2 or FIG. 3,polynucleotides encoding about amino acid 90 to about amino acid 100 ofthe 205P1B5 protein shown in FIG. 2 or FIG. 3, in increments of about 10amino acids, ending at the carboxyl terminal amino acid set forth inFIG. 2 or FIG. 3. Accordingly polynucleotides encoding portions of theamino acid sequence (of about 10 amino acids), of amino acids 100through the carboxyl terminal amino acid of the 205P1B5 protein areembodiments of the invention. Wherein it is understood that eachparticular amino acid position discloses that position plus or minusfive amino acid residues.

Polynucleotides encoding relatively long portions of the 205P1B5 proteinare also within the scope of the invention. For example, polynucleotidesencoding from about amino acid 1 (or 20 or 30 or 40 etc.) to about aminoacid 20, (or 30, or 40 or 50 etc.) of the 205P1B500 protein shown inFIG. 2 or FIG. 3 can be generated by a variety of techniques well knownin the art. These polynucleotide fragments can include any portion ofthe 205P1B5 sequence as shown in FIG. 2 or FIG. 3.

Additional illustrative embodiments of the invention disclosed hereininclude 205P1B5 polynucleotide fragments encoding one or more of thebiological motifs contained within the 205P1B5 protein sequence,including one or more of the motif-bearing subsequences of the 205P1B5protein set forth in Tables V-XVIII. In another embodiment, typicalpolynucleotide fragments of the invention encode one or more of theregions of 205P1B5 that exhibit homology to a known molecule. In anotherembodiment of the invention, typical polynucleotide fragments can encodeone or more of the 205P1B5 N-glycosylation sites, cAMP andcGMP-dependent protein kinase phosphorylation sites, casein kinase IIphosphorylation sites or N-myristoylation site and amidation sites.

II.A.) Uses of 205P1B5 Polynucleotides

II.A.1.) Monitoring of Genetic Abnormalities

The polynucleotides of the preceding paragraphs have a number ofdifferent specific uses. The human 205P1B5 gene maps to the chromosomallocation set forth in Example 3. For example, because the 205P1B5 genemaps to this chromosome, polynucleotides that encode different regionsof the 205P1B5 protein are used to characterize cytogeneticabnormalities of this chromosomal locale, such as abnormalities that areidentified as being associated with various cancers. In certain genes, avariety of chromosomal abnormalities including rearrangements have beenidentified as frequent cytogenetic abnormalities in a number ofdifferent cancers (see e.g. Krajinovic et al., Mutat. Res. 382(3-4):81-83 (1998); Johansson et al., Blood 86(10): 3905-3914 (1995) andFinger et al., P.N.A.S. 85(23): 9158-9162 (1988)). Thus, polynucleotidesencoding specific regions of the 205P1B5 protein provide new tools thatcan be used to delineate, with greater precision than previouslypossible, cytogenetic abnormalities in the chromosomal region thatencodes 205P1B5 that may contribute to the malignant phenotype. In thiscontext, these polynucleotides satisfy a need in the art for expandingthe sensitivity of chromosomal screening in order to identify moresubtle and less common chromosomal abnormalities (see e.g. Evans et al.,Am. J. Obstet. Gynecol. 171(4): 1055-1057 (1994)).

Furthermore, as 205P1B5 was shown to be highly expressed in prostate andother cancers, 205P1B5 polynucleotides are used in methods assessing thestatus of 205P1B5 gene products in normal versus cancerous tissues.Typically, polynucleotides that encode specific regions of the 205P1B5protein are used to assess the presence of perturbations (such asdeletions, insertions, point mutations, or alterations resulting in aloss of an antigen etc.) in specific regions of the 205P1B5 gene, suchas such regions containing one or more motifs. Exemplary assays includeboth RT-PCR assays as well as single-strand conformation polymorphism(SSCP) analysis (see, e.g., Marrogi et al., J. Cutan. Pathol. 26(8):369-378 (1999), both of which utilize polynucleotides encoding specificregions of a protein to examine these regions within the protein.

II.A.2.) Antisense Embodiments

Other specifically contemplated nucleic acid related embodiments of theinvention disclosed herein are genomic DNA, cDNAs, ribozymes, andantisense molecules, as well as nucleic acid molecules based on analternative backbone, or including alternative bases, whether derivedfrom natural sources or synthesized, and include molecules capable ofinhibiting the RNA or protein expression of 205P1B5. For example,antisense molecules can be RNAs or other molecules, including peptidenucleic acids (PNAs) or non-nucleic acid molecules such asphosphorothioate derivatives, that specifically bind DNA or RNA in abase pair-dependent manner. A skilled artisan can readily obtain theseclasses of nucleic acid molecules using the 205P1B5 polynucleotides andpolynucleotide sequences disclosed herein.

Antisense technology entails the administration of exogenousoligonucleotides that bind to a target polynucleotide located within thecells. The term “antisense” refers to the fact that sucholigonucleotides are complementary to their intracellular targets, e.g.,205P1B5. See for example, Jack Cohen, Oligodeoxynucleotides, AntisenseInhibitors of Gene Expression, CRC Press, 1989; and Synthesis 1:1-5(1988). The 205P1B5 antisense oligonucleotides of the present inventioninclude derivatives such as S-oligonucleotides (phosphorothioatederivatives or S-oligos, see, Jack Cohen, supra), which exhibit enhancedcancer cell growth inhibitory action. S-oligos (nucleosidephosphorothioates) are isoelectronic analogs of an oligonucleotide(O-oligo) in which a nonbridging oxygen atom of the phosphate group isreplaced by a sulfur atom The S-oligos of the present invention can beprepared by treatment of the corresponding O-oligos with3H-1,2-benzodithiol-3-one-1,1-dioxide, which is a sulfur transferreagent See Iyer, R. P. et al., J. Org. Chem. 55:4693-4698 (1990); andIyer, R. P. et al., J. Am. Chem. Soc. 112:1253-1254 (1990). Additional205P1B5 antisense oligonucleotides of the present invention includemorpholino antisense oligonucleotides known in the art (see, e.g.,Partridge et al., 1996, Antisense & Nucleic Acid Drug Development 6:169-175).

The 205P1B5 antisense oligonucleotides of the present inventiontypically can be RNA or DNA that is complementary to and stablyhybridizes with the first 100 5′ codons or last 100 3′ codons of the205P1B5 genomic sequence or the corresponding mRNA. Absolutecomplementarity is not required, although high degrees ofcomplementarity are preferred. Use of an oligonucleotide complementaryto this region allows for the selective hybridization to 205P1B5 mRNAand not to mRNA specifying other regulatory subunits of protein kinase.In one embodiment, 205P1B5 antisense oligonucleotides of the presentinvention are 15 to 30-mer fragments of the antisense DNA molecule thathave a sequence that hybridizes to 205P1B5 mRNA. Optionally, 205P1B5antisense oligonucleotide is a 30-mer oligonucleotide that iscomplementary to a region in the first 10 5′ codons or last 10 3′ codonsof 205P1B5. Alternatively, the antisense molecules are modified toemploy ribozymes in the inhibition of 205P1B5 expression, see, e.g., L.A. Couture & D. T. Stinchcomb; Trends Genet. 12: 510-515 (1996).

II.A3.) Primers and Primer Pairs

Further specific embodiments of this nucleotides of the inventioninclude primers and primer pairs, which allow the specific amplificationof polynucleotides of the invention or of any specific parts thereof,and probes that selectively or specifically hybridize to nucleic acidmolecules of the invention or to any part thereof. Probes can be labeledwith a detectable marker, such as, for example, a radioisotope,fluorescent compound, bioluminescent compound, a chemiluminescentcompound, metal chelator or enzyme. Such probes and primers are used todetect the presence of a 205P1B5 polynucleotide in a sample and as ameans for detecting a cell expressing a 205P1B5 protein.

Examples of such probes include polypeptides comprising all or part ofthe human 205P1B5 cDNA sequence shown in FIG. 2. Examples of primerpairs capable of specifically amplifying 205P1B5 mRNAs are alsodescribed in the Examples. As will be understood by the skilled artisan,a great many different primers and probes can be prepared based on thesequences provided herein and used effectively to amplify and/or detecta 205P1B5 mRNA.

The 205P1B5 polynucleotides of the invention are useful for a variety ofpurposes, including but not limited to their use as probes and primersfor the amplification and/or detection of the 205P1B5 gene(s), mRNA(s),or fragments thereof; as reagents for the diagnosis and/or prognosis ofprostate cancer and other cancers; as coding sequences capable ofdirecting the expression of 205P1B5 polypeptides; as tools formodulating or inhibiting the expression of the 205P1B5 gene(s) and/ortranslation of the 205P1B5 transcript(s); and as therapeutic agents.

The present invention includes the use of any probe as described hereinto identify and isolate a 205P1B5 or 205P1B5 related nucleic acidsequence from a naturally occurring source, such as humans or othermammals, as well as the isolated nucleic acid sequence per se, whichwould comprise all or most of the sequences found in the probe used.

II.A.4.) Isolation of 205P1B5 Encoding Nucleic Acid Molecules

The 205P1B5 cDNA sequences described herein enable the isolation ofother polynucleotides encoding 205P1B5 gene product(s), as well as theisolation of polynucleotides encoding 205P1B5 gene product homologs,alternatively spliced isoforms, allelic variants, and mutant forms ofthe 205P1B5 gene product as well as polynucleotides that encode analogsof 205P1B5-related proteins. Various molecular cloning methods that canbe employed to isolate full length cDNAs encoding an 205P1B5 gene arewell known (see, for example, Sambrook J. et al., Molecular Cloning: ALaboratory Manual, 2d edition, Cold Spring Harbor Press, New York, 1989;Current Protocols in Molecular Biology. Ausubel et al., Eds., Wiley andSons, 1995). For example, lambda phage cloning methodologies can beconveniently employed, using commercially available cloning systems(e.g., Lambda ZAP Express, Stratagene). Phage clones containing 205P1B5gene cDNAs can be identified by probing with a labeled 205P1B5 cDNA or afragment thereof. For example, in one embodiment, the 205P1B5 cDNA (FIG.2) or a portion thereof can be synthesized and used as a probe toretrieve overlapping and full-length cDNAs corresponding to a 205P1B5gene. The 205P1B5 gene itself can be isolated by screening genomic DNAlibraries, bacterial artificial chromosome libraries (BACs), yeastartificial chromosome libraries (YACs), and the like, with 205P1B5 DNAprobes or primers.

II.A.5.) Recombinant Nucleic Acid Molecules and Host-Vector Systems

The invention also provides recombinant DNA or RNA molecules containingan 205P1B5 polynucleotide, a fragment, analog or homologue thereof,including but not limited to phages, plasmids, phagemids, cosmids, YACs,BACs, as well as various viral and non-viral vectors well known in theart, and cells transformed or transfected with such recombinant DNA orRNA molecules. Methods for generating such molecules are well known(see, for example, Sambrook et al., 1989, supra).

The invention further provides a host-vector system comprising arecombinant DNA molecule containing a 205P1B5 polynucleotide, fragment,analog or homologue thereof within a suitable prokaryotic or eukaryotichost cell. Examples of suitable eukaryotic host cells include a yeastcell, a plant cell, or an animal cell, such as a mammalian cell or aninsect cell (e.g., a baculovirus-infectible cell such as an Sf9 orHighFive cell). Examples of suitable mammalian cells include variousprostate cancer cell lines such as DU145 and TsuPr1, other transfectableor transducible prostate cancer cell lines, primary cells (PrEC), aswell as a number of mammalian cells routinely used for the expression ofrecombinant proteins (e.g., COS, CHO, 293, 293T cells). Moreparticularly, a polynucleotide comprising the coding sequence of 205P1B5or a fragment, analog or homolog thereof can be used to generate 205P1B5proteins or fragments thereof using any number of host-vector systemsroutinely used and widely known in the art.

A wide range of host-vector systems suitable for the expression of205P1B5 proteins or fragments thereof are available, see for example,Sambrook et al., 1989, supra; Current Protocols in Molecular Biology,1995, supra). Preferred vectors for mammalian expression include but arenot limited to pcDNA 3.1 myc-His-tag (Invitrogen) and the retroviralvector pSRαtkneo (Muller et al., 1991, MCB 11:1785). Using theseexpression vectors, 205P1B5 can be expressed in several prostate cancerand non-prostate cell lines, including for example 293, 293T, rat-1, NIH3T3 and TsuPr1. The host-vector systems of the invention are useful forthe production of a 205P1B5 protein or fragment thereof. Suchhost-vector systems can be employed to study the functional propertiesof 205P1B5 and 205P1B5 mutations or analogs.

Recombinant human 205P1B5 protein or an analog or homolog or fragmentthereof can be produced by mammalian cells transfected with a constructencoding a 205P1B5-related nucleotide. For example, 293T cells can betransfected with an expression plasmid encoding 205P1B5 or fragment,analog or homolog thereof, the 205P1B5 or related protein is expressedin the 293T cells, and the recombinant 205P1B5 protein is isolated usingstandard purification methods (e.g., affinity purification usinganti-205P1B5 antibodies). In another embodiment, a 205P1B5 codingsequence is subcloned into the retroviral vector pSRαMSVtkneo and usedto infect various mammalian cell lines, such as NIH 3T3, TsuPr1, 293 andrat-1 in order to establish 205P1B5 expressing cell lines. Various otherexpression systems well known in the art can also be employed.Expression constructs encoding a leader peptide joined in frame to the205P1B5 coding sequence can be used for the generation of a secretedform of recombinant 205P1B5 protein.

As discussed herein, redundancy in the genetic code permits variation in205P1B5 gene sequences. In particular, it is known in the art thatspecific host species often have specific codon preferences, and thusone can adapt the disclosed sequence as preferred for a desired host.For example, preferred analog codon sequences typically have rare codons(i.e., codons having a usage frequency of less than about 20% in knownsequences of the desired host) replaced with higher frequency codons.Codon preferences for a specific species are calculated, for example, byutilizing codon usage tables available on the INTERNET such as at worldwide web URL dna.affrc.go.jp/-nakamura/codon.html.

Additional sequence modifications are known to enhance proteinexpression in a cellular host. These include elimination of sequencesencoding spurious polyadenylation signals, exon/intron splice sitesignals, transposon-like repeats, and/or other such well-characterizedsequences that are deleterious to gene expression. The GC content of thesequence is adjusted to levels average for a given cellular host, ascalculated by reference to known genes expressed in the host cell. Wherepossible, the sequence is modified to avoid predicted hairpin secondarymRNA structures. Other useful modifications include the addition of atranslational initiation consensus sequence at the start of the openreading frame, as described in Kozak, Mol. Cell Biol., 9:5073-5080(1989). Skilled artisans understand that the general rule thateukaryotic ribosomes initiate translation exclusively at the 5′ proximalAUG codon is abrogated only under rare conditions (see, e.g., Kozak PNAS92(7): 2662-2666, (1995) and Kozak NAR 15(20): 8125-8148 (1987)).

III.) 205P1B5-Related Proteins

Another aspect of the present invention provides 205P1B5-relatedproteins. Specific embodiments of 205P1B5 proteins comprise apolypeptide having all or part of the amino acid sequence of human205P1B5 as shown in FIG. 2 or FIG. 3. Alternatively, embodiments of265P1B5 proteins comprise variant, homolog or analog polypeptides thathave alterations in the amino acid sequence of 205P1B5 shown in FIG. 2or FIG. 3.

In general, naturally occurring allelic variants of human 205P1B5 sharea high degree of structural identity and homology (e.g., 90% or morehomology). Typically, allelic variants of the 205P1B5 protein containconservative amino acid substitutions within the 205P1B5 sequencesdescribed herein or contain a substitution of an amino acid from acorresponding position in a homologue of 205P1B5. One class of 205P1B5allelic variants are proteins that share a high degree of homology withat least a small region of a particular 205P1B5 amino acid sequence, butfurther contain a radical departure from the sequence, such as anon-conservative substitution, truncation, insertion or frame shift Incomparisons of protein sequences, the terms, similarity, identity, andhomology each have a distinct meaning as appreciated in the field ofgenetics. Moreover, orthology and paralogy can be important conceptsdescribing the relationship of members of a given protein family in oneorganism to the members of the same family in other organisms.

Amino acid abbreviations are provided in Table II. Conservative aminoacid substitutions can frequently be made in a protein without alteringeither the conformation or the function of the protein. Proteins of theinvention can comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15conservative substitutions. Such changes include substituting any ofisoleucine (I), valine (V), and leucine (L) for any other of thesehydrophobic amino acids; aspartic acid (D) for glutamic acid (E) andvice versa; glutamine (Q) for asparagine (N) and vice versa; and serine(S) for threonine (T) and vice versa. Other substitutions can also beconsidered conservative, depending on the environment of the particularamino acid and its role in the three dimensional structure of theprotein. For example, glycine (G) and alanine (A) can frequently beinterchangeable, as can alanine (A) and valine (V). Methionine (M),which is relatively hydrophobic, can frequently be interchanged withleucine and isoleucine, and sometimes with valine. Lysine (K) andarginine (R) are frequently interchangeable in locations in which thesignificant feature of the amino acid residue is its charge and thediffering pK's of these two amino acid residues are not significantStill other changes can be considered “conservative” in particularenvironments (see, e.g. Table III herein; pages 13-15 “Biochemistry”2^(nd) ED. Lubert Stryer ed. (Stanford University); Henikoff et al.,PNAS 1992 Vol. 89 10915-10919; Lei et al., J. Biol. Chem. 1995 May 19;270(20):11882-6).

Embodiments of the invention disclosed herein include a wide variety ofart-accepted variants or analogs of 205P1B5 proteins such aspolypeptides having amino acid insertions, deletions and substitutions.205P1B5 variants can be made using methods known in the art such assite-directed mutagenesis, alanine scanning, and PCR mutagenesis.Site-directed mutagenesis (Carter et al., Nucl. Acids Res., 13:4331(1986); Zoller et al., Nucl. Acids Res., 10:6487 (1987)), cassettemutagenesis (Wells et al., Gene, 34:315 (1985)), restriction selectionmutagenesis (Wells et al., Philos. Trans. R. Soc. London SerA, 317:415(1986)) or other known techniques can be performed on the cloned DNA toproduce the 205P1B5 variant DNA.

Scanning amino acid analysis can also be employed to identify one ormore amino acids along a contiguous sequence that is involved in aspecific biological activity such as a protein-protein interaction.Among the preferred scanning amino acids are relatively small, neutralamino acids. Such amino acids include alanine, glycine, serine, andcysteine. Alanine is typically a preferred scanning amino acid amongthis group because it eliminates the side-chain beyond the beta-carbonand is less likely to alter the main-chain conformation of the variant.Alanine is also typically preferred because it is the most common aminoacid. Further, it is frequently found in both buried and exposedpositions (Creighton, The Proteins, (W. H. Freeman & Co., N.Y.);Chothia, J. Mol. Biol., 150:1 (1976)). If alanine substitution does notyield adequate amounts of variant, an isosteric amino acid can be used.

As defined herein, 205P1B5 variants, analogs or homologs, have thedistinguishing attribute of having at least one epitope that is “crossreactive” with a 205P1B5 protein having the amino acid sequence of SEQID NO: 703. As used in this sentence, “cross reactive” means that anantibody or T cell that specifically binds to an 205P1B5 variant alsospecifically binds to the 205P1B5 protein having the amino acid sequenceof SEQ ID NO: 703. A polypeptide ceases to be a variant of the proteinshown in SEQ ID NO: 703 when it no longer contains any epitope capableof being recognized by an antibody or T cell that specifically binds tothe 205P1B5 protein. Those skilled in the art understand that antibodiesthat recognize proteins bind to epitopes of varying size, and a groupingof the order of about four or five amino acids, contiguous or not, isregarded as a typical number of amino acids in a minimal epitope. See,e.g., Nair et al., J. Immunol. 2000 165(12): 6949-6955; Hebbes et al.,Mol. Immunol. (1989) 26(9):865-73; Schwartz et al., J. Immunol. (1985)135(4):2598-608.

Another class of 205P1B5-related protein variants share 70%, 75%, 80%,85% or 90% or more similarity with the amino acid sequence of SEQ ID NO:703 or a fragment thereof. Another specific class of 205P1B5 proteinvariants or analogs comprise one or more of the 205P1B5 biologicalmotifs described herein or presently known in the art. Thus, encompassedby the present invention are analogs of 205P1B5 fragments (nucleic oramino acid) that have altered functional (e.g. immunogenic) propertiesrelative to the starting fragment. It is to be appreciated that motifsnow or which become part of the art are to be applied to the nucleic oramino acid sequences of FIG. 2 or FIG. 3.

As discussed herein, embodiments of the claimed invention includepolypeptides containing less than the full amino acid sequence of the205P1B5 protein shown in FIG. 2 or FIG. 3. For example, representativeembodiments of the invention comprise peptides/proteins having any 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more contiguous amino acids of the205P1B5 protein shown in FIG. 2 or FIG. 3.

Moreover, representative embodiments of the invention disclosed hereininclude polypeptides consisting of about amino acid 1 to about aminoacid 10 of the 205P1B5 protein shown in FIG. 2 or FIG. 3, polypeptidesconsisting of about amino acid 10 to about amino acid 20 of the 205P1B5protein shown in FIG. 2 or FIG. 3, polypeptides consisting of aboutamino acid 20 to about amino acid 30 of the 205P1B5 protein shown inFIG. 2 or FIG. 3, polypeptides consisting of about amino acid 30 toabout amino acid 40 of the 205P1B5 protein shown in FIG. 2 or FIG. 3,polypeptides consisting of about amino acid 40 to about amino acid 50 ofthe 205P1B5 protein shown in FIG. 2 or FIG. 3, polypeptides consistingof about amino acid 50 to about amino acid 60 of the 205P1B5 proteinshown in FIG. 2 or FIG. 3, polypeptides consisting of about amino acid60 to about amino acid 70 of the 205P1B5 protein shown in FIG. 2 or FIG.3, polypeptides consisting of about amino acid 70 to about amino acid 80of the 205P1B5 protein shown in FIG. 2 or FIG. 3, polypeptidesconsisting of about amino acid 80 to about amino acid 90 of the 205P1B5protein shown in FIG. 2 or FIG. 3, polypeptides consisting of aboutamino acid 90 to about amino acid 100 of the 205P1B5 protein shown inFIG. 2 or FIG. 3, etc. throughout the entirety of the 205P1B5 amino acidsequence. Moreover, polypeptides consisting of about amino acid 1 (or 20or 30 or 40 etc.) to about amino acid 20, (or 130, or 140 or 150 etc.)of the 205P1B5 protein shown in FIG. 2 or FIG. 3 are embodiments of theinvention. It is to be appreciated that the starting and stoppingpositions in this paragraph refer to the specified position as well asthat position plus or minus 5 residues.

205P1B5-related proteins are generated using standard peptide synthesistechnology or using chemical cleavage methods well known in the art.Alternatively, recombinant methods can be used to generate nucleic acidmolecules that encode a 205P1B5-related protein. In one embodiment,nucleic acid molecules provide a means to generate defined fragments ofthe 205P1B5 protein (or variants, homologs or analogs thereof).

III.A.) Motif-Bearing Protein Embodiments

Additional illustrative embodiments of the invention disclosed hereininclude 205P1B5 polypeptides comprising the amino acid residues of oneor more of the biological motifs contained within the 205P1B5polypeptide sequence set forth in FIG. 2 or FIG. 3. Various motifs areknown in the art, and a protein can be evaluated for the presence ofsuch motifs by a number of publicly available Internet sites (see, e.g.,URL addresses: pfam.wustl.edu/;searchlauncher.bcm.tmc.edu/seq-search/struc-predict.htmlpsort.ims.u-tokvo.ac.jp/; world wide web URL cbs.dtu.dk/; world wide webURL ebi.ac.uk/interpro/scan.html; world wide web URLexpasy.c/tools/scnpsitl.html; Epimatrix™ and Epimer™, Brown University,world wide web URLbrown.edu/Research/TB-HIV_Lab/epimatri/epimatrix.html; and BIMAS,bimas.dcrt.nih.gov/.).

Motif bearing subsequences of the 205P1B5 protein are set forth andidentified in Table XIX. Table XX sets forth several frequentlyoccurring motifs based on pfam searches (see URL addresspfamwustl.edu/). The columns of Table XX list (1) motif nameabbreviation, (2) percent identity found amongst the different member ofthe motif family, (3) motif name or description and (4) most commonfunction; location information is included if the motif is relevant forlocation.

Polypeptides comprising one or more of the 205P1B5 motifs discussedabove are useful in elucidating the specific characteristics of amalignant phenotype in view of the observation that the 205P1B5 motifsdiscussed above are associated with growth dysregulation and because205P1B5 is overexpressed in certain cancers (See, e.g., Table I). Caseinkinase II, cAMP and camp-dependent protein kinase, and Protein Kinase C,for example, are enzymes known to be associated with the development ofthe malignant phenotype (see e.g. Chen et al., Lab Invest., 78(2):165-174 (1998); Gaiddon et al., Endocrinology 136(10): 4331-4338 (1995);Hall et al., Nucleic Acids Research 24(6): 1119-1126 (1996); Peterzielet al., Oncogene 18(46): 6322-6329 (1999) and O'Brian, Oncol. Rep. 5(2):305-309 (1998)). Moreover, both glycosylation and myristoylation areprotein modifications also associated with cancer and cancer progression(see e.g. Dennis et al., Biochem. Biophys. Acta 1473(1):21-34 (1999);Raju et al., Exp. Cell. Res. 235(1): 145-154 (1997)). Amidation isanother protein modification also associated with cancer and cancerprogression (see e.g. Treston et al., J. Natl. Cancer Inst. Monogr.(13): 169-175 (1992)).

In another embodiment, proteins of the invention comprise one or more ofthe immunoreactive epitopes identified in accordance with art-acceptedmethods, such as the peptides set forth in Tables V-XVIII. CTL epitopescan be determined using specific algorithms to identify peptides withinan 205P1B5 protein that are capable of optimally binding to specifiedHLA alleles (e.g., Table IV; EphnaTrix™ and Epimer™, Brown University,URL world wide web URLbrown.edu/Research/TB-HIV_Lab/epimatrix/epimatrix.html; and BIMAS, URLbimas.dcrt.nih.gov/.) Moreover, processes for identifying peptides thathave sufficient binding affinity for HLA molecules and which arecorrelated with being immunogenic epitopes, are well known in the art,and are carried out without undue experimentation. In addition,processes for identifying peptides that are inmunogenic epitopes, arewell known in the art, and are carried out without undue experimentationeither in vitro or in vivo.

Also known in the art are principles for creating analogs of suchepitopes in order to modulate immunogenicity. For example, one beginswith an epitope that bears a CTL or HTL motif (see, e.g., the HLA ClassI and HLA Class II motifs/supermotifs of Table IV). The epitope isanalogued by substituting out an amino acid at one of the specifiedpositions, and replacing it with another amino acid specified for thatposition. For example, one can substitute out a deleterious residue infavor of any other residue, such as a preferred residue as defined inTable IV; substitute a less-preferred residue with a preferred residueas defined in Table IV; or substitute an originally-occurring preferredresidue with another preferred residue as defined in Table IV.Substitutions can occur at primary anchor positions or at otherpositions in a peptide; see, e.g., Table IV.

A variety of references reflect the art regarding the identification andgeneration of epitopes in a protein of interest as well as analogsthereof. See, for example, WO 9733602 to Chesnut et al.; Sette,Immunogenetics 1999 50(3-4): 201-212; Sette et al., J. Immunol. 2001166(2): 1389-1397; Sidney et al., Hum. Immunol. 1997 58(1): 12-20; Kondoet al., Immunogenetics 1997 45(4): 249-258; Sidney et al., J. Immunol.1996 157(8): 3480-90; and Falk et al., Nature 351: 290-6 (1991); Hunt etal., Science 255:1261-3 (1992); Parker et al., J. Immunol. 149:3580-7(1992); Parker et al., J. Immunol. 152:163-75 (1994)); Kast et al., 1994152(8): 3904-12; Borras-Cuesta et al., Hum. Immunol. 2000 61(3):266-278; Alexander et al., J. Immunol. 2000 164(3); 164(3): 1625-1633;Alexander et al., PMID: 7895164, U: 95202582; O'Sullivan et al., J.Immunol. 1991 147(8): 2663-2669; Alexander et al., Immunol. 1994 1(9):751-761 and Alexander et al., Immunol. Res. 1998 18(2): 79-92.

Related embodiments of the inventions include polypeptides comprisingcombinations of the different motifs set forth in Table XIX, and/or, oneor more of the predicted CTL epitopes of Table V through Table XVIII,and/or, one or more of the T cell binding motifs known in the art.Preferred embodiments contain no insertions, deletions or substitutionseither within the motifs or the intervening sequences of thepolypeptides. In addition, embodiments which include a number of eitherN-terminal and/or C-terminal amino acid residues on either side of thesemotifs may be desirable (to, for example, include a greater portion ofthe polypeptide architecture in which the motif is located). Typicallythe number of N-terminal and/or C-terminal amino acid residues on eitherside of a motif is between about 1 to about 100 amino acid residues,preferably 5 to about 50 amino acid residues.

205P1B5-related proteins are embodied in many forms, preferably inisolated form A purified 205P1B5 protein molecule will be substantiallyfree of other proteins or molecules that impair the binding of 205P1B5to antibody, T cell or other ligand. The nature and degree of isolationand purification will depend on the intended use. Embodiments of a205P1B5-related proteins include purified 205P1B5-related proteins andfunctional, soluble 205P1B5-related proteins. In one embodiment, afunctional soluble 205P1B5 protein or fragment thereof retains theability to be bound by antibody, T cell or other ligand.

The invention also provides 205P1B5 proteins comprising biologicallyactive fragments of the 205P1B5 amino acid sequence shown in FIG. 2 orFIG. 3. Such proteins exhibit properties of the 205P1B5 protein, such asthe ability to elicit the generation of antibodies that specificallybind an epitope associated with the 205P1B5 protein; to be bound by suchantibodies; to elicit the activation of HTL or CTL; and/or, to berecognized by HTL or CTL. 205P1B5-related polypeptides that containparticularly interesting structures can be predicted and/or identifiedusing various analytical techniques well known in the art, including,for example, the methods of Chou-Fasman, Garnier-Robson, Kyte-Doolittle,Eisenberg, Karplus-Schultz or Jameson-Wolf analysis, or on the basis ofimmunogenicity. Fragments that contain such structures are particularlyuseful in generating subunit-specific anti-205P1B5 antibodies, or Tcells or in identifying cellular factors that bind to 205P1B5.

CTL epitopes can be determined using specific algorithms to identifypeptides within an 205P1B5 protein that are capable of optimally bindingto specified HLA alleles (e.g., by using the SYFPEITHI site at WorldWide Web URL syfpeithi.bmi-heidelberg.com/; the listings in TableIV(A)-(E); Epimatrix™ and Epimer™, Brown University, URL (world wide webURL brown.edu/Research/TB-IIIV_Lab/epimatrix/epimatrix.html); and BIMAS,URL bimas.dcrt.nih.gov/). Illustrating this, peptide epitopes from205P1B5 that are presented in the context of human MHC class I moleculesHLA-A1, A2, A3, A11, A24, B7 and B35 were predicted (Tables V-XVIII).Specifically, the complete amino acid sequence of the 205P1B5 proteinwas entered into the HLA Peptide Motif Search algorithm found in theBioinformatics and Molecular Analysis Section (BIMAS) web site listedabove. The HLA peptide motif search algorithm was developed by Dr. KenParker based on binding of specific peptide sequences in the groove ofHLA Class I molecules, in particular HLA-A2 (see, e.g., Falk et al.,Nature 351: 290-6 (1991); Hunt et al., Science 255:1261-3 (1992); Parkeret al., J. Immunol. 149:3580-7 (1992); Parker et al., J. Immunol.152:163-75 (1994)). This algorithm allows location and ranking of 8-mer,9-mer, and 10-mer peptides from a complete protein sequence forpredicted binding to HLA-A2 as well as numerous other HLA Class Imolecules. Many HLA class I binding peptides are 8-, 9-, 10 or 11-mers.For example, for class I HLA-A2, the epitopes preferably contain aleucine (L) or methionine (M) at position 2 and a valine (V) or leucine(L) at the C-terminus (see, e.g., Parker et al., J. Immunol. 149:3580-7(1992)). Selected results of 205P1B5 predicted binding peptides areshown in Tables V-XVII herein. In Tables V-XVIII, the top 50 rankingcandidates, 9-mers and 10-mers, for each family member are shown alongwith their location, the amino acid sequence of each specific peptide,and an estimated binding score. The binding score corresponds to theestimated half time of dissociation of complexes containing the peptideat 37° C. at pH 6.5. Peptides with the highest binding score arepredicted to be the most tightly bound to HLA Class I on the cellsurface for the greatest period of time and thus represent the bestimmunogenic targets for T-cell recognition.

Actual binding of peptides to an HLA allele can be evaluated bystabilization of HLA expression on the antigen-processing defective cellline T2 (see, e.g., Xue et al., Prostate 30:73-8 (1997) and Peshwa etal., Prostate 36:129-38 (1998)). Immunogenicity of specific peptides canbe evaluated an vitro by stimulation of CD8+ cytotoxic T lymphocytes(CTL) in the presence of antigen presenting cells such as dendriticcells.

It is to be appreciated that every epitope predicted by the BIMAS site,Epimer™ and Epimatrix™ sites, or specified by the HLA class I or classII motifs available in the art or which become part of the art such asset forth in Table IV (or determined using World Wide Web site URLsyfpeithi.bmi-heidelberg.com/) are to be “applied” to the 205P1B5protein. As used in this context “applied” means that the 205P1B5protein is evaluated, e.g., visually or by computer-based patternsfinding methods, as appreciated by those of skill in the relevant art.Every subsequence of the 205P1B5 of 8, 9, 10, or 11 amino acid residuesthat bears an HLA Class I motif, or a subsequence of 9 or more aminoacid residues that bear an HLA Class II motif are within the scope ofthe invention.

III.B.) Expression of 205P1B5-Related Proteins

In an embodiment described in the examples that follow, 205P1B5 can beconveniently expressed in cells (such as 293T cells) transfected with acommercially available expression vector such as a CMV-driven expressionvector encoding 205P1B5 with a C-terminal 6×His and MYC tag(pcDNA3.1/mycHIS, Invitrogen or Tag5, GenHunter Corporation, NashvilleTenn.). The Tag5 vector provides an IgGK secretion signal that can beused to facilitate the production of a secreted 205P1B5 protein intransfected cells. The secreted HIS-tagged 205P1B5 in the culture mediacan be purified, e.g., using a nickel column using standard techniques.

III.C.) Modifications of 205P1B5-Related Proteins

Modifications of 205P1B5-related proteins such as covalent modificationsare included within the scope of this invention. One type of covalentmodification includes reacting targeted amino acid residues of a 205P1B5polypeptide with an organic derivatizing agent that is capable ofreacting with selected side chains or the N- or C-terminal residues ofthe 205P1B5. Another type of covalent modification of the 205P1B5polypeptide included within the scope of this invention comprisesaltering the native glycosylation pattern of a protein of the invention.Another type of covalent modification of 205P1B5 comprises linking the205P1B5 polypeptide to one of a variety of nonproteinaceous polymers,e.g., polyethylene glycol (PEG), polypropylene glycol, orpolyoxyalkylenes, in the manner set forth in U.S. Pat. Nos. 4,640,835;4,496,689; 4,301,144; 4,670,417; 4,791,192 or 4,179,337.

The 205P1B5-related proteins of the present invention can also bemodified to form a chimeric molecule comprising 205P1B5 fused toanother, heterologous polypeptide or amino acid sequence. Such achimeric molecule can be synthesized chemically or recombinantly. Achimeric molecule can have a protein of the invention fused to anothertumor-associated antigen or fragment thereof. Alternatively, a proteinin accordance with the invention can comprise a fusion of fragments ofthe 205P1B5 sequence (amino or nucleic acid) such that a molecule iscreated that is not, through its length, directly homologous to theamino or nucleic acid sequences shown in FIG. 2 or FIG. 3. Such achimeric molecule can comprise multiples of the same subsequence of205P1B5. A chimeric molecule can comprise a fusion of a 205P1B5-relatedprotein with a polyhistidine epitope tag, which provides an epitope towhich immobilized nickel can selectively bind, with cytolines or withgrowth factors. The epitope tag is generally placed at the amino- orcarboxyl-terminus of the 205P1B5. In an alternative embodiment, thechimeric molecule can comprise a fusion of a 205P1B5-related proteinwith an immunoglobulin or a particular region of an immunoglobulin. Fora bivalent form of the chimeric molecule (also referred to as an“immunoadhesin”), such a fusion could be to the Fc region of an IgGmolecule. The Ig fusions preferably include the substitution of asoluble (transmembrane domain deleted or inactivated) form of a 205P1B5polypeptide in place of at least one variable region within an Igmolecule. In a preferred embodiment, the immunoglobulin fusion includesthe hinge, CH2 and CH3, or the hinge, CH1, CH2 and CH3 regions of anIgGI molecule. For the production of immunoglobulin fusions see, e.g.,U.S. Pat. No. 5,428,130 issued Jun. 27, 1995.

III.D.) Uses of 205P1B5-Related Proteins

The proteins of the invention have a number of different specific uses.As 205P1B5 is highly expressed in prostate and other cancers,205P1B5-related proteins are used in methods that assess the status of205P1B5 gene products in normal versus cancerous tissues, therebyelucidating the malignant phenotype. Typically, polypeptides fromspecific regions of the 205P1B5 protein are used to assess the presenceof perturbations (such as deletions, insertions, point mutations etc.)in those regions (such as regions containing one or more motifs).Exemplary assays utilize antibodies or T cells targeting 205P1B5-relatedproteins comprising the amino acid residues of one or more of thebiological motifs contained within the 205P1B5 polypeptide sequence inorder to evaluate the characteristics of this region in normal versuscancerous tissues or to elicit an immune response to the epitope.Alternatively, 205P1B5-related proteins that contain the amino acidresidues of one or more of the biological motifs in the 205P1B5 proteinare used to screen for factors that interact with that region of205P1B5.

205P1B5 protein fragments/subsequences are particularly useful ingenerating and characterizing domain specific antibodies (e.g.,antibodies recognizing an extracellular or intracellular epitope of an205P1B5 protein), for identifying agents or cellular factors that bindto 205P1B5 or a particular structural domain thereof, and in varioustherapeutic and diagnostic contexts, including but not limited todiagnostic assays, cancer vaccines and methods of preparing suchvaccines.

Proteins encoded by the 205P1B5 genes, or by analogs, homologs orfragments thereof, have a variety of uses, including but not limited togenerating antibodies and in methods for identifying ligands and otheragents and cellular constituents that bind to an 205P1B5 gene productAntibodies raised against an 205P1B5 protein or fragment thereof areuseful in diagnostic and prognostic assays, and imaging methodologies inthe management of human cancers characterized by expression of 205P1B5protein, such as those listed in Table I. Such antibodies can beexpressed intracellularly and used in methods of treating patients withsuch cancers. 205P1B5-related nucleic acids or proteins are also used ingenerating HTL or CTL responses.

Various immunological assays useful for the detection of 205P1B5proteins are used, including but not limited to various types ofradioimmunoassays, enzyme-linked immunosorbent assays (ELISA),enzyme-linked immunofluorescent assays (ELIFA), immunocytochemicalmethods, and the like. Antibodies can be labeled and used asimmunological imaging reagents capable of detecting 205P1B5expressingcells (e.g., in radioscintigraphic imaging methods). 205P1B5 proteinsare also particularly useful in generating cancer vaccines, as furtherdescribed herein.

IV.) 205P1B5 Antibodies

Another aspect of the invention provides antibodies that bind to205P1B5-related proteins. Preferred antibodies specifically bind to a205P1B5-related protein and do not bind (or bind weakly) to peptides orproteins that are not 205P1B5-related proteins. For example, antibodiesbind 205P1B5 can bind 205P1B5-related proteins such as the homologs oranalogs thereof.

205P1B5 antibodies of the invention are particularly useful in prostatecancer diagnostic and prognostic assays, and imaging methodologies.Similarly, such antibodies are useful in the treatment, diagnosis,and/or prognosis of other cancers, to the extent 205P1B5 is alsoexpressed or overexpressed in these other cancers. Moreover,intracellularly expressed antibodies (e.g., single chain antibodies) aretherapeutically useful in treating cancers in which the expression of205P1B5 is involved, such as advanced or metastatic prostate cancers.

The invention also provides various immunological assays useful for thedetection and quantification of 205P1B5 and mutant 205P1B5-relatedproteins. Such assays can comprise one or more 205P1B5 antibodiescapable of recognizing and binding a 205P1B5-related protein, asappropriate. These assays are performed within various immunologicalassay formats well known in the art, including but not limited tovarious types of radioimmunoassays, enzyme-linked immunosorbent assays(ELISA), enzyme linked immunofluorescent assays (ELIFA), and the like.

Immunological non-antibody assays of the invention also comprise T cellimmunogenicity assays (inhibitory or stimulatory) as well as majorhistocompatibility complex (MHC) binding assays.

In addition, immunological imaging methods capable of detecting prostatecancer and other cancers expressing 205P1B5 are also provided by theinvention, including but not limited to radioscintigraphic imagingmethods using labeled 205P1B5 antibodies. Such assays are clinicallyuseful in the detection, monitoring, and prognosis of 205P1B5 expressingcancers such as prostate cancer. 205P1B5 antibodies are also used inmethods r purifying a 205P1B5-related protein and for isolating 205P1B5homologues and related molecules. For example, a method of purifying a205P1B5-related protein comprises incubating an 205P1B5 antibody, whichhas been coupled to a solid matrix, with a lysate or other solutioncontaining a 205P1B5-related protein under conditions that permit the205P1B5 antibody to bind to the 205P1B5-related protein; washing thesolid matrix to eliminate impurities; and eluting the 205P1B5-relatedprotein from the coupled antibody. Other uses of the 205P1B5 antibodiesof the invention include generating anti-idiotypic antibodies that mimicthe 205P1B5 protein.

Various methods for the preparation of antibodies are well known in theart. For example, antibodies can be prepared by immunizing a suitablemammalian host using a 205P1B5-related protein, peptide, or fragment, inisolated or immunoconjugated form (Antibodies: A Laboratory Manual, CSHPress, Eds., Harlow, and Lane (1988); Harlow, Antibodies, Cold SpringHarbor Press, N.Y. (1989)). In addition, fusion proteins of 205P1B5 canalso be used, such as a 205P1B5 GST-fusion protein. In a particularembodiment, a GST fusion protein comprising all or most of the aminoacid sequence of FIG. 2 or FIG. 3 is produced, then used as an immunogento generate appropriate antibodies. In another embodiment, a205P1B5-related protein is synthesized and used as an immunogen.

In addition, naked DNA immunization techniques known in the art are used(with or without purified 205P1B5-related protein or 205P1B5 expressingcells) to generate an immune response to the encoded immunogen (forreview, see Donnelly et al., 1997, Ann. Rev. Immunol. 15: 61768).

The amino acid sequence of 205P1B5 as shown in FIG. 2 or FIG. 3 can beanalyzed to select specific regions of the 205P1B5 protein forgenerating antibodies. For example, hydrophobicity and hydrophilicityanalyses of the 205P1B5 amino acid sequence are used to identifyhydrophilic regions in the 205P1B5 structure. Regions of the 205P1B5protein that show immunogenic structure, as well as other regions anddomains, can readily be identified using various other methods known inthe ark such as Chou-Fasman, Garnier-Robson, Kyte-Doolittle, Eisenberg,Karplus-Schultz or Jameson-Wolf analysis. Thus, each region identifiedby any of these programs or methods is within the scope of the presentinvention. Methods for the generation of 205P1B5 antibodies are furtherillustrated by way of the examples provided herein. Methods forpreparing a protein or polypeptide for use as an immunogen are wellknown in the art. Also well known in the art are methods for preparingimmunogenic conjugates of a protein with a carrier, such as BSA, KLH orother carrier protein. In some circumstances, direct conjugation using,for example, carbodiimide reagents are used; in other instances linkingreagents such as those supplied by Pierce Chemical Co., Rockford, Ill.,are effective. Administration of a 205P1B5 immunogen is often conductedby injection over a suitable time period and with use of a suitableadjuvant, as is understood in the art. During the immunization schedule,titers of antibodies can be taken to determine adequacy of antibodyformation.

205P1B5 monoclonal antibodies can be produced by various means wellknown in the art. For example, immortalized cell lines that secrete adesired monoclonal antibody are prepared using the standard hybridomatechnology of Kohler and Milstein or modifications that immortalizeantibody-producing B cells, as is generally known Immortalized celllines that secrete the desired antibodies are screened by immunoassay inwhich the antigen is a 205P1B5-related protein. When the appropriateimmortalized cell culture is identified, the cells can be expanded andantibodies produced either from in vitro cultures or from ascites fluid.

The antibodies or fragments of the invention can also be produced, byrecombinant means. Regions that bind specifically to the desired regionsof the 205P1B5 protein can also be produced in the context of chimericor complementarity determining region (CDR) gifted antibodies ofmultiple species origin. Humanized or human 205P1B5 antibodies can alsobe produced, and are preferred for use in therapeutic contexts. Methodsfor humming mine and other non-human antibodies, by substituting one ormore of the non-human antibody CDRs for corresponding human antibodysequences, are well known (see for example, Jones et al., 1986, Nature321: 522-525; Riechmann et al., 1988, Nature 332: 323-327; Verhoeyen etal., 1988, Science 239: 1534-1536). See also, Carter et al., 1993, Proc.Natl. Acad. Sci. USA 89: 4285 and Sims et al., 1993, J. Immunol. 151:2296.

Methods for producing fully human monoclonal antibodies include phagedisplay and transgenic methods (for review, see Vaughan et al., 1998,Nature Biotechnology 16: 535-539). Fully human 205P1B5 monoclonalantibodies can be generated using cloning technologies employing largehuman Ig gene combinatorial libraries (i.e., phage display) (Griffithsand Hoogenboom, Building an in vitro immune system human antibodies fromphage display libraries. In: Protein Engineering of Antibody Moleculesfor Prophylactic and Therapeutic Applications in Man, Clark, M. (Ed.),Nottingham Academic, pp 45-64 (1993); Burton and Barbas, HumanAntibodies from combinatorial libraries. Id, pp 65-82). Fully human205P1B5 monoclonal antibodies can also be produced using transgenic miceengineered to contain human immunoglobulin gene loci as described in PCTPatent Application WO98/24893, Kucherlapati and Jakobovits et al.,published Dec. 3, 1997 (see also, Jakobovits, 1998, Exp. Opin. Invest.Drugs 7(4): 607-614; U.S. Pat. No. 6,162,963 issued 19 Dec. 2000; U.S.Pat. No. 6,150,584 issued 12 Nov. 2000; and, U.S. Pat. No. 6,114,598issued 5 Sep. 2000). This method avoids the in vitro manipulationrequired with phage display technology and efficiently produces highaffinity authentic human antibodies.

Reactivity of 205P1B5 antibodies with an 205P1B5-related protein can beestablished by a number of well known means, including Western blot,immunoprecipitation, ELISA, and FACS analyses using, as appropriate,205P1B5-related proteins, 205P1B5-expressing cells or extracts thereof.A 205P1B5 antibody or fragment thereof can be labeled with a detectablemarker or conjugated to a second molecule. Suitable detectable markersinclude, but are not limited to, a radioisotope, a fluorescent compound,a bioluminescent compound, chemiluminescent compound, a metal chelatoror an enzyme. Further, bi-specific antibodies specific for two or more205P1B5 epitopes are generated using methods generally known in the artHomodimeric antibodies can also be generated by cross-linking techniquesknown in the art (e.g., Wolff et al., Cancer Res. 53: 2560-2565).

V.) 205P1B5 Cellular Immune Responses

The mechanism by which T cells recognize antigens has been delineated.Efficacious peptide epitope vaccine compositions of the invention inducea therapeutic or prophylactic immune responses in very broad segments ofthe world-wide population. For an understanding of the value andefficacy of compositions of the invention that induce cellular immuneresponses, a brief review of immunology-related technology is provided

A complex of an HLA molecule and a peptidic antigen acts as the ligandrecognized by HLA-restricted T cells (Buus, S. et al., Cell 47:1071,1986; Babbitt, B. P. et al., Nature 317:359, 1985; Townsend, A. andBodmer, H., Annu. Rev. Immunol. 7:601, 1989; Germain, R. N., Annu. Rev.Immunol. 11:403, 1993). Through the study of single amino acidsubstituted antigen analogs and the sequencing of endogenously bound,naturally processed peptides, critical residues that correspond tomotifs required for specific binding to HLA antigen molecules have beenidentified and are set forth in Table IV (see also, e.g., Southwood, etal., J. Immunol. 160:3363, 1998; Rammensee, et al., Immunogenetics41:178, 1995; Rammensee et al., SYFPEITHI, access via World Wide Web atURL syfpeithi.bmi-heidelberg.com/; Sette, A. and Sidney, J. Curr. Opin.Immunol. 10:478, 1998; Engelhard, V. H., Curr. Opin. Immunol. 6:13,1994; Sette, A. and Grey, H. M., Curr. Opin. Immunol. 4:79, 1992;Sinigaglia, F. and Hammer, J. Curr. Biol. 6:52, 1994; Ruppert et al.,Cell 74:929-937, 1993; Kondo et al., J. Immunol. 155:4307-4312, 1995;Sidney et al., J. Immunol. 157:3480-3490, 1996; Sidney et al., HumanImmunol. 45:79-93, 1996; Sette, A. and Sidney, J. Immunogenetics 1999November; 50(34):201-12, Review).

Furthermore, x-ray crystallographic analyses of HLA-peptide complexeshave revealed pockets within the peptide binding cleft/groove of HLAmolecules which accommodate, in an allele-specific mode, residues borneby peptide ligands; these residues in turn determine the HLA bindingcapacity of the peptides in which they are present (See, e.g., Madden,D. R. Annu. Rev. Immunol. 13:587, 1995; Smith, et al., Immunity 4:203,1996; Fremont et al., Immunity 8:305, 1998; Stern et al., Structure2:245, 1994; Jones, E. Y. Curr. Opin. Immunol. 9:75, 1997; Brown, J. H.et al., Nature 364:33, 1993; Guo, H. C. et al., Proc. Natl. Acad. Sci.USA 90:8053, 1993; Guo, H. C. et al., Nature 360:364, 1992; Silver, M.L. et al., Nature 360:367, 1992; Matsumura, M. et al., Science 257:927,1992; Madden et al., Cell 70:1035, 1992; Fremont, D. H. et al., Science257:919, 1992; Saper, M. A., Bjorkman, P. J. and Wiley, D. C., J. Mol.Biol. 219:277, 1991.)

Accordingly, the definition of class I and class II allele-specific HLAbinding motifs, or class I or class II supermotifs allows identificationof regions within a protein that are correlated with binding toparticular HLA antigen(s).

Thus, by a process of HLA motif identification, candidates forepitope-based vaccines have been identified; such candidates can befurther evaluated by HLA-peptide binding assays to determine bindingaffinity and/or the time period of association of the epitope and itscorresponding HLA molecule. Additional confirmatory work can beperformed to select, amongst these vaccine candidates, epitopes withpreferred characteristics in terms of population coverage, and/orimmunogenicity.

Various strategies can be utilized to evaluate cellular immunogenicity,including:

1) Evaluation of primary T cell cultures from normal individuals (see,e.g., Wentworth, P. A. et al., Mol. Immunol. 32:603, 1995; Celis, E. etal., Proc. Natl. Acad. Sci. USA 91:2105, 1994; Tsai, V. et al., J.Immunol. 158:1796, 1997; Kawashima, I. et al., Human Immunol. 59:1,1998). This procedure involves the stimulation of peripheral bloodlymphocytes (PBL) from normal subjects with a test peptide in thepresence of antigen presenting cells in vitro over a period of severalweeks. T cells specific for the peptide become activated during thistime and are detected using, e.g., a lymphokine- or ⁵¹Cr-release assayinvolving peptide sensitized target cells.

2) Immunization of HLA transgenic mice (see, e.g., Wentworth, P. A. etal., J. Immunol. 26:97, 1996; Wentworth, P. A. et al., Int. Immunol.8:651, 1996; Alexander, J. et al., J. Immunol. 159:4753, 1997). Forexample, in such methods peptides in incomplete Freund's adjuvant areadministered subcutaneously to HLA transgenic mice. Several weeksfollowing immunization, splenocytes are removed and cultured in vitro inthe presence of test peptide for approximately one week.Peptide-specific T cells are detected using, e.g., a ⁵¹Cr-release assayinvolving peptide sensitized target cells and target cells expressingendogenously generated antigen.

3) Demonstration of recall T cell responses from immune individuals whohave been either effectively vaccinated and/or from chronically illpatients (see, e.g., Rehermann, B. et al., J. Exp. Med. 181:1047, 1995;Doolan, D. L. et al., Immunity 7:97, 1997; Bertoni, R et al., J. Clin.Invest. 100:503, 1997; Threlkeld, S. C. et al., J. Immunol. 159:1648,1997; Diepolder, H. M. et al., J. Virol. 71:6011, 1997). Accordingly,recall responses are detected by culturing PBL from subjects that havebeen exposed to the antigen due to disease and thus have generated animmune response “naturally”, or from patients who were vaccinatedagainst the antigen. PBL from subjects are cultured in vitro for 1-2weeks in the presence of test peptide plus antigen presenting cells(APC) to allow activation of “memory” T cells, as compared to “naive” Tcells. At the end of the culture period, T cell activity is detectedusing assays including ⁵¹Cr release involving peptide-sensitizedtargets, T cell proliferation, or lymphokine release.

VI.) 205P1B5 Transgenic Animals

Nucleic acids that encode a 205P1B5-related protein can also be used togenerate either transgenic animals or “knock out” animals which, inturn, are useful in the development and screening of therapeuticallyuseful reagents. In accordance with established techniques, cDNAencoding 205P1B5 can be used to clone genomic DNA that encodes 205P1B5.The cloned genomic sequences can then be used to generate transgenicanimals containing cells that express DNA that encode 205P1B5. Methodsfor generating transgenic animals, particularly animals such as mice orrats, have become conventional in the art and are described, forexample, in U.S. Pat. No. 4,736,866 issued 12 Apr. 1988, and U.S. Pat.No. 4,870,009 issued 26 Sep. 1989. Typically, particular cells would betargeted for 205P1B5 transgene incorporation with tissue-specificenhancers.

Transgenic animals that include a copy of a transgene encoding 205P1B5can be used to examine the effect of increased expression of DNA thatencodes 205P1B5. Such animals can be used as tester animals for reagentsthought to confer protection from, for example, pathological conditionsassociated with its overexpression. In accordance with this aspect ofthe invention, an animal is treated with a reagent and a reducedincidence of a pathological condition, compared to untreated animalsthat bear the transgene, would indicate a potential therapeuticintervention for the pathological condition.

Alternatively, non-human homologues of 205P1B5 can be used to constructa 205P1B5 “knock out” animal that has a defective or altered geneencoding 205P1B5 as a result of homologous recombination between theendogenous gene encoding 205P1B5 and altered genomic DNA encoding205P1B5 introduced into an embryonic cell of the animal. For example,cDNA that encodes 205P1B5 can be used to clone genomic DNA encoding205P1B5 in accordance with established techniques. A portion of thegenomic DNA encoding 205P1B5 can be deleted or replaced with anothergene, such as a gene encoding a selectable marker that can be used tomonitor integration. Typically, several kilobases of unaltered flankingDNA (both at the 5′ and 3′ ends) are included in the vector (see, e.g.,Thomas and Capecchi, Cell, 51:503 (1987) for a description of homologousrecombination vectors). The vector is introduced into an embryonic stemcell line (e.g., by electroporation) and cells in which the introducedDNA has homologously recombined with the endogenous DNA are selected(see, e.g., Li et al., Cell 69:915 (1992)). The selected cells are theninjected into a blastocyst of an animal (e.g., a mouse or rat) to formaggregation chimeras (see, e.g., Bradley, in Teratocarcinomas andEmbryonic Stem Cells: A Practical Approach, E. J. Robertson, ed. (IRL,Oxford, 1987), pp. 113-152). A chimeric embryo can then be implantedinto a suitable pseudopregnant female foster animal, and the embryobrought to term to create a “knock out” animal. Progeny harboring thehomologously recombined DNA in their germ cells can be identified bystandard techniques and used to breed animals in which all cells of theanimal contain the homologously recombined DNA. Knock out animals can becharacterized, for example, for their ability to defend against certainpathological conditions or for their development of pathologicalconditions due to absence of the 205P1B5 polypeptide.

VII.) Methods for the Detection of 205P1B5

Another aspect of the present invention relates to methods for detecting205P1B5 polynucleotides and 205P1B5-related proteins, as well as methodsfor identifying a cell that expresses 205P1B5. The expression profile of205P1B5 makes it a diagnostic marker for metastasized disease.Accordingly, the status of 205P1B5 gene products provides informationuseful for predicting a variety of factors including susceptibility toadvanced stage disease, rate of progression, and/or tumoraggressiveness. As discussed in detail herein, the status of 205P1B5gene products in patient samples can be analyzed by a variety protocolsthat are well known in the art including immunohistochemical analysis,the variety of Northern blotting techniques including in situhybridization, RT-PCR analysis (for example on laser capturemicrodissected samples), Western blot analysis and tissue arrayanalysis.

More particularly, the invention provides assays for the detection of205P1B5 polynucleotides in a biological sample, such as serum, bone,prostate, and other tissues, urine, semen, cell preparations, and thelike. Detectable 205P1B5 polynucleotides include, for example, a 205P1B5gene or fragment thereof, 205P1B5 mRNA, alternative splice variant205P1B5 mRNAs, and recombinant DNA or RNA molecules that contain a205P1B5 polynucleotide. A number of methods for amplifying and/ordetecting the presence of 205P1B5 polynucleotides are well known in theart and can be employed in the practice of this aspect of the invention.

In one embodiment, a method for detecting an 205P1B5 mRNA in abiological sample comprises producing cDNA from the sample by reversetranscription using at least one primer; amplifying the cDNA so producedusing an 205P1B5 polynucleotides as sense and antisense primers toamplify 205P1B5 cDNAs therein; and detecting the presence of theamplified 205P1B5 cDNA. Optionally, the sequence of the amplified205P1B5 cDNA can be determined.

In another embodiment, a method of detecting a 205P1B5 gene in abiological sample comprises first isolating genomic DNA from the sample;amplifying the isolated genomic DNA using 205P1B5 polynucleotides assense and antisense primers; and detecting the presence of the amplified205P1B5 gene. Any number of appropriate sense and antisense probecombinations can be designed from the nucleotide sequence provided forthe 205P1B5 (FIG. 2) and used for this purpose.

The invention also provides assays for detecting the presence of an205P1B5 protein in a tissue or other biological sample such as serum,semen, bone, prostate, urine, cell preparations, and the like. Methodsfor detecting a 205P1B5-related protein are also well known and include,for example, immunoprecipitation, immunohistochemical analysis, Westernblot analysis, molecular binding assays, ELISA, ELIFA and the like. Forexample, a method of detecting the presence of a 205P1B5-related proteinin a biological sample comprises first contacting the sample with a205P1B5 antibody, a 205P1B5-reactive fragment thereof, or a recombinantprotein containing an antigen binding region of a 205P1B5 antibody; andthen detecting the binding of 205P1B5-related protein in the sample.

Methods for identifying a cell that expresses 205P1B5 are also withinthe scope of the invention In one embodiment, an assay for identifying acell that expresses a 205P1B5 gene comprises detecting the presence of205P1B5 mRNA in the cell. Methods for the detection of particular mRNAsin cells are well known and include, for example, hybridization assaysusing complementary DNA probes (such as in situ hybridization usinglabeled 205P1B5 riboprobes, Northern blot and related techniques) andvarious nucleic acid amplification assays (such as RT-PCR usingcomplementary primers specific for 205P1B5, and other amplification typedetection methods, such as, for example, branched DNA, SISBA, TMA andthe like). Alternatively, an assay for identifying a cell that expressesa 205P1B5 gene comprises detecting the presence of 205P1B5-relatedprotein in the cell or secreted by the cell. Various methods for thedetection of proteins are well known in the art and are employed for thedetection of

205P1B5-related proteins and cells that express 205P1B5-relatedproteins. 205P1B5 expression analysis is also useful as a tool foridentifying and evaluating agents that modulate 205P1B5 gene expression.For example, 205P1B5 expression is significantly upregulated in prostatecancer, and is expressed in cancers of the tissues listed in Table I.Identification of a molecule or biological agent that inhibits 205P1B5expression or over-expression in cancer cells is of therapeutic value.For example, such an agent can be identified by using a screen thatquantifies 205P1B5 expression by RT-PCR, nucleic acid hybridization orantibody binding.

VIII.) Methods for Monitoring the Status of 205P1B5-Related Genes andTheir Products

Oncogenesis is known to be a multistep process where cellular growthbecomes progressively dysregulated and cells progress from a normalphysiological state to precancerous and then cancerous states (see,e.g., Alers, et al., Lab Invest. 77(5): 437-438 (1997) and Isaacs etal., Cancer Surv. 23: 19-32 (1995)). In this context, examining abiological sample for evidence of dysregulated cell growth (such asaberrant 205P1B5 expression in cancers) allows for early detection ofsuch aberrant physiology, before a pathologic state such as cancer hasprogressed to a stage that therapeutic options are more limited and orthe prognosis is worse. In such examinations, the status of 205P1B5 in abiological sample of interest can be compared, for example, to thestatus of 205P1B5 in a corresponding normal sample (e.g. a sample fromthat individual or alternatively another individual that is not affectedby a pathology). An alteration in the status of 205P1B5 in thebiological sample (as compared to the normal sample) provides evidenceof dysregulated cellular growth. In addition to using a biologicalsample that is not affected by a pathology as a normal sample, one canalso use a predetermined normative value such as a predetermined normallevel of mRNA expression (see, e.g., Grever et al., J. Comp. Neurol.1996 Dec. 9;376(2):306-14 and U.S. Pat. No. 5,837,501) to compare205P1B5 status in a sample.

The tern “status” in this context is used according to its art acceptedmeaning and refers to the condition or state of a gene and its products.Typically, skilled artisans use a number of parameters to evaluate thecondition or state of a gene and its products. These include, but arenot limited to the location of expressed gene products (including thelocation of 205P1B5 expressing cells) as well as the level, andbiological activity of expressed gene products (such as 205P1B5 mRNA,polynucleotides and polypeptides). Typically, an alteration in thestatus of 205P1B5 comprises a change in the location of 205P1B5 and/or205P1B5 expressing cells and/or an increase in 205P1B5 mRNA and/orprotein expression.

205P1B5 status in a sample can be analyzed by a number of means wellknown in the art, including without limitation, immunohistochemicalanalysis, in situ hybridization, RT-PCR analysis on laser capturemicro-dissected samples, Western blot analysis, and tissue arrayanalysis. Typical protocols for evaluating the status of the 205P1B5gene and gene products are found, for example in Ausubel et al. eds.,1995, Current Protocols In Molecular Biology, Units 2 (NorthernBlotting), 4 (Southern Blotting), 15 (Immunoblotting) and 18 (PCRAnalysis). Thus, the status of 205P1B5 in a biological sample isevaluated by various methods utilized by skilled artisans including, butnot limited to genomic Southern analysis (to examine, for exampleperturbations in the 205P1B5 gene), Northern analysis and/or PCRanalysis of 205P1B5 mRNA (to examine, for example alterations in thepolynucleotide sequences or expression levels of 205P1B5 mRNAs), and,Western and/or immunohistochemical analysis (to examine, for examplealterations in polypeptide sequences, alterations in polypeptidelocalization within a sample, alterations in expression levels of205P1B5 proteins and/or associations of 205P1B5 proteins withpolypeptide binding partners). Detectable 205P1B5 polynucleotidesinclude, for example, a 205P1B5 gene or fragment thereof, 205P1B5 mRNA,alternative splice variants, 205P1B5 mRNAs, and recombinant DNA or RNAmolecules containing a 205P1B5 polynucleotide.

The expression profile of 205P1B5 makes it a diagnostic marker for localand/or metastasized disease, and provides information on the growth oroncogenic potential of a biological sample. In particular, the status of205P1B5 provides information useful for predicting susceptibility toparticular disease stages, progression, and/or tumor aggressiveness. Theinvention provides methods and assays for determining 205P1B5 status anddiagnosing cancers that express 205P1B5, such as cancers of the tissueslisted in Table I. For example, because 205P1B5 mRNA is so highlyexpressed in prostate and other cancers relative to normal prostatetissue, assays that evaluate the levels of 205P1B5 mRNA transcripts orproteins in a biological sample can be used to diagnose a diseaseassociated with 205P1B5 dysregulation, and can provide prognosticinformation useful in defining appropriate therapeutic options.

The expression status of 205P1B5 provides information including thepresence, stage and location of dysplastic, precancerous and cancerouscells, predicting susceptibility to various stages of disease, and/orfor gauging tumor aggressiveness. Moreover, the expression profile makesit useful as an imaging reagent for metastasized disease. Consequently,an aspect of the invention is directed to the various molecularprognostic and diagnostic methods for examining the status of 205P1B5 inbiological samples such as those from individuals suffering from, orsuspected of suffering from a pathology characterized by dysregulatedcellular growth, such as cancer.

As described above, the status of 205P1B5 in a biological sample can beexamined by a number of well-known procedures in the art. For example,the status of 205P1B5 in a biological sample taken from a specificlocation in the body can be examined by evaluating the sample for thepresence or absence of 205P1B5expressing cells (e.g. those that express205P1B5 mRNAs or proteins). This examination can provide evidence ofdysregulated cellular growth, for example, when 205P1B5-expressing cellsare found in a biological sample that does not normally contain suchcells (such as a lymph node), because such alterations in the status of205P1B5 in a biological sample are often associated with dysregulatedcellular growth. Specifically, one indicator of dysregulated cellulargrowth is the metastases of cancer cells from an organ of origin (suchas the prostate) to a different area of the body (such as a lymph node).In this context, evidence of dysregulated cellular growth is importantfor example because occult lymph node metastases can be detected in asubstantial proportion of patients with prostate cancer, and suchmetastases are associated with known predictors of disease progression(see, e.g., Murphy et al., Prostate 42(4): 315-317 (2000);Su et al.,Semin. Surg. Oncol. 18(1): 17-28 (2000) and Freeman et al., J. Urol 1995August 154(2 Pt 1):474-8).

In one aspect, the invention provides methods for monitoring 205P1B5gene products by determining the status of 205P1B5 gene productsexpressed by cells from an individual suspected of having a diseaseassociated with dysregulated cell growth (such as hyperplasia or cancer)and then comparing the status so determined to the status of 205P1B5gene products in a corresponding normal sample. The presence of aberrant205P1B5 gene products in the test sample relative to the normal sampleprovides an indication of the presence of dysregulated cell growthwithin the cells of the individual.

In another aspect, the invention provides assays useful in determiningthe presence of cancer in an individual, comprising detecting asignificant increase in 205P1B5 mRNA or protein expression in a testcell or tissue sample relative to expression levels in the correspondingnormal cell or tissue. The presence of 205P1B5 mRNA can, for example, beevaluated in tissue samples including but not limited to those listed inTable I. The presence of significant 205P1B5 expression in any of thesetissues is useful to indicate the emergence, presence and/or severity ofa cancer, since the corresponding normal tissues do not express 205P1B5mRNA or express it at lower levels.

In a related embodiment, 205P1B5 status is determined at the proteinlevel rather than at the nucleic acid level. For example, such a methodcomprises determining the level of 205P1B5 protein expressed by cells ina test tissue sample and comparing the level so determined to the levelof 205P1B5 expressed in a corresponding normal sample. In oneembodiment, the presence of 205P1B5 protein is evaluated, for example,using immunohistochemical methods. 205P1B5 antibodies or bindingpartners capable of detecting 205P1B5 protein expression are used in avariety of assay formats well known in the art for this purpose.

In a further embodiment, one can evaluate the status of 205P1B5nucleotide and amino acid sequences in a biological sample in order toidentify perturbations in the structure of these molecules. Theseperturbations can include insertions, deletions, substitutions and thelike. Such evaluations are useful because perturbations in thenucleotide and amino acid sequences are observed in a large number ofproteins associated with a growth dysregulated phenotype (see, e.g.,Marrogi et al., 1999, J. Cutan. Pathol. 26(8):369-378). For example, amutation in the sequence of 205P1B5 may be indicative of the presence orpromotion of a tumor. Such assays therefore have diagnostic andpredictive value where a mutation in 205P1B5 indicates a potential lossof function or increase in tumor growth.

A wide variety of assays for observing perturbations m nucleotide andamino acid sequences are well known in the art. For example, the sizeand structure of nucleic acid or amino acid sequences of 205P1B5 geneproducts are observed by the Northern, Southern, Western, PCR and DNAsequencing protocols discussed herein. In addition, other methods forobserving perturbations in nucleotide and amino acid sequences such assingle strand conformation polymorphism analysis are well known in theart (see, e.g., U.S. Pat. No. 5,382,510 issued 7 Sep. 1999, and U.S.Pat. No. 5,952,170 issued 17 Jan. 1995).

Additionally, one can examine the methylation status of the 205P1B5 genein a biological sample. Aberrant demethylation and/or hypermethylationof CpG islands in gene 5′ regulatory regions frequently occurs inimmortalized and transformed cells, and can result in altered expressionof various genes. For example, promoter hypermethylation of the pi-classglutathione S-transferase (a protein expressed in normal prostate butnot expressed in>90% of prostate carcinomas) appears to permanentlysilence transcription of this gene and is the most frequently detectedgenomic alteration in prostate carcinomas (De Marzo et al., Am. J.Pathol. 155(6): 1985-1992 (1999)). In addition, this alteration ispresent in at least 70% of cases of high-grade prostatic intraepithelialneoplasia (PIN) (Brooks et al., Cancer Epidemiol. Biomarkers Prev.,1998, 7:531-536). In another example, expression of the LAGE-I tumorspecific gene (which is not expressed in normal prostate but isexpressed in 25-50% of prostate cancers) is induced by deoxy-azacytidinein lymphoblastoid cells, suggesting that tumoral expression is due todemethylation (Lethe et al., Int. J. Cancer 76(6): 903-908 (1998)). Avariety of assays for examining methylation status of a gene are wellknown in the art For example, one can utilize, in Southern hybridizationapproaches, methylation-sensitive restriction enzymes which cannotcleave sequences that contain methylated CpG sites to assess themethylation status of CpG islands. In addition, MSP (methylationspecific PCR) can rapidly profile the methylation status of all the CpGsites present in a CpG island of a given gene. This procedure involvesinitial modification of DNA by sodium bisulfite (which will convert allunmethylated cytosines to uracil) followed by amplification using priersspecific for methylated versus unmethylated DNA. Protocols involvingmethylation interference can also be found for example in CurrentProtocols In Molecular Biology, Unit 12, Frederick M. Ausubel et al.eds., 1995.

Gene amplification is an additional method for assessing the status of205P1B5. Gene amplification is measured in a sample directly, forexample, by conventional Southern blotting or Northern blotting toquantitate the transcription of mRNA (Thomas, 1980, Proc. Natl. Acad.Sci. USA, 77:5201-5205), dot blotting (DNA analysis), or in situhybridization, using an appropriately labeled probe, based on thesequences provided herein. Alternatively, antibodies are employed thatrecognize specific duplexes, including DNA duplexes, RNA duplexes, andDNA-RNA hybrid duplexes or DNA-protein duplexes. The antibodies in turnare labeled and the assay carried out where the duplex is bound to asurface, so that upon the formation of duplex on the surface, thepresence of antibody bound to the duplex can be detected.

Biopsied tissue or peripheral blood can be conveniently assayed for thepresence of cancer cells using for example, Northern, dot blot or RT-PCRanalysis to detect 205P1B5 expression. The presence of RT-PCRamplifiable 205P1B5 mRNA provides an indication of the presence ofcancer. RT-PCR assays are well known in the art RT-PCR detection assaysfor tumor cells in peripheral blood are currently being evaluated foruse in the diagnosis and management of a number of human solid tumors.In the prostate cancer field, these include RT-PCR assays for thedetection of cells expressing PSA and PSM (Verkaik et al., 1997, Urol.Res. 25:373-384; Ghossein et al., 1995, J. Clin. Oncol. 13:1195-2000;Heston et al., 1995, Clin. Chem. 41:1687-1688).

A further aspect of the invention is an assessment of the susceptibilitythat an individual has for developing cancer. In one embodiment, amethod for predicting susceptibility to cancer comprises detecting205P1B5 mRNA or 205P1B5 protein in a tissue sample, its presenceindicating susceptibility to cancer, wherein the degree of 205P1B5 mRNAexpression correlates to the degree of susceptibility. In a specificembodiment, the presence of 205P1B5 in prostate or other tissue isexamined, with the presence of 205P1B5 in the sample providing anindication of prostate cancer susceptibility (or the emergence orexistence of a prostate tumor). Similarly, one can evaluate theintegrity 205P1B5 nucleotide and amino acid sequences in a biologicalsample, in order to identify perturbations in the structure of thesemolecules such as insertions, deletions, substitutions and the like. Thepresence of one or more perturbations in 205P1B5 gene products in thesample is an indication of cancer susceptibility (or the emergence orexistence of a tumor).

The invention also comprises methods for gauging tumor aggressiveness.In one embodiment, a method for gauging aggressiveness of a tumorcomprises determining the level of 205P1B5 mRNA or 205P1B5 proteinexpressed by tumor cells, comparing the level so determined to the levelof 205P1B5 mRNA or 205P1B5 protein expressed in a corresponding normalissue taken from the same individual or a normal tissue referencesample, wherein the degree of 205P1B5 mRNA or 205P1B5 protein expressionin the tumor sample relative to the normal sample indicates the degreeof aggressiveness. In a specific embodiment, aggressiveness of a tumoris evaluated by determining the extent to which 205P1B5 is expressed inthe tumor cells, with higher expression levels indicating moreaggressive tumors. Another embodiment is the evaluation of the integrityof 205P1B5 nucleotide and amino acid sequences in a biological sample,in order to identify perturbations in the structure of these moleculessuch as insertions, deletions, substitutions and the like. The presenceof one or more perturbations indicates more aggressive tumors.

Another embodiment of the invention is directed to methods for observingthe progression of a malignancy in an individual over time. In oneembodiment, methods for observing the progression of a malignancy in anindividual over time comprise determining the level of 205P1B5 mRNA or205P1B5 protein expressed by cells in a sample of the tumor, comparingthe level so determined to the level of 205P1B5 mRNA or 205P1B5 proteinexpressed in an equivalent tissue sample taken from the same individualat a different time, wherein the degree of 205P1B5 mRNA or 205P1B5protein expression in the tumor sample overtime provides information onthe progression of the cancer. In a specific embodiment, the progressionof a cancer is evaluated by determining 205P1B5 expression in the tumorcells over time, where increased expression over time indicates aprogression of the cancer. Also, one can evaluate the integrity 205P1B5nucleotide and amino acid sequences in a biological sample in order toidentify perturbations in the structure of these molecules such asinsertions, deletions, substitutions and the like, where the presence ofone or more perturbations indicates a progression of the cancer.

The above diagnostic approaches can be combined with any one of a widevariety of prognostic and diagnostic protocols known in the art. Forexample, another embodiment of the invention is directed to methods forobserving a coincidence between the expression of 205P1B5 gene and205P1B5 gene products (or perturbations in 205P1B5 gene and 205P1B5 geneproducts) and a factor that is associated with malignancy, as a meansfor diagnosing and prognosticating the status of a tissue sample. A widevariety of factors associated with malignancy can be utilized, such asthe expression of genes associated with malignancy (e.g. PSA, PSCA andPSM expression for prostate cancer etc.) as well as gross cytologicalobservations (see, e.g., Bocking et al., 1984, Anal. Quant. Cytol.6(2):74-88; Epstein, 1995, Hum. Pathol. 26(2):223-9; Thorson et al.,1998, Mod. Pathol. 11(6):543-51; Baisden et al., 1999, Am. J. Surg.Pathol. 23(8):918-24). Methods for observing a coincidence between theexpression of 205P1B5 gene and 205P1B5 gene products (or perturbationsin 205P1B5 gene and 205P1B5 gene products) and another factor that isassociated with malignancy are useful, for example, because the presenceof a set of specific factors that coincide with disease providesinformation crucial for diagnosing and prognosticating the status of atissue sample.

In one embodiment, methods for observing a coincidence between theexpression of 205P1B5 gene and 205P1B5 gene products (or perturbationsin 205P1B5 gene and 205P1B5 gene products) and another factor associatedwith malignancy entails detecting the overexpression of 205P1B5 mRNA orprotein in a tissue sample, detecting the overexpression of PSA mRNA orprotein in a tissue sample (or PSCA or PSM expression), and observing acoincidence of 205P1B5 mRNA or protein and PSA mRNA or proteinoverexpression (or PSCA or PSM expression). In a specific embodiment,the expression of 205P1B5 and PSA mRNA in prostate tissue is examined,where the coincidence of 205P1B5 and PSA mRNA overexpression in thesample indicates the existence of prostate cancer, prostate cancersusceptibility or the emergence or status of a prostate tumor.

Methods for detecting and quantifying the expression of 205P1B5 mRNA orprotein are described herein, and standard nucleic acid and proteindetection and quantification technologies are well known in the art.Standard methods for the detection and quantification of 205P1B5 mRNAinclude in situ hybridization using labeled 205P1B5 riboprobes, Northernblot and related techniques using 205P1B5 polynucleotide probes, RT-PCRanalysis using primers specific for 205P1B5, and other amplificationtype detection methods, such as, for example, branched DNA, SISBA, TMAand the like. In a specific embodiment, semi-quantitative RT-PCR is usedto detect and quantify 205P1B5 mRNA expression. Any number of primerscapable of amplifying 205P1B5 can be used for this purpose, includingbut not limited to the various primer sets specifically describedherein. In a specific embodiment, polyclonal or monoclonal antibodiesspecifically reactive with the wild-type 205P1B5 protein can be used inan immumohistochemical assay of biopsied issue.

IX) Identification of Molecules that Interact with 205P1B5

The 205P1B5 protein and nucleic acid sequences disclosed herein allow askilled artisan to identify proteins, small molecules and other agentsthat interact with 205P1B5, as well as pathways activated by 205P1B5 viaany one of a variety of art accepted protocols. For example, one canutilize one of the so-called interaction trap systems (also referred toas the “two-hybrid assay”). In such systems, molecules interact andreconstitute a transcription factor which directs expression of areporter gene, whereupon the expression of the reporter gene is assayed.Other systems identify protein-protein interactions in vivo throughreconstitution of a eukaryotic transcriptional activator, see, e.g.,U.S. Pat. No. 5,955,280 issued 21 Sep. 1999, U.S. Pat. No. 5,925,523issued 20 Jul. 1999, U.S. Pat. No. 5,846,722 issued 8 Dec. 1998 and U.S.Pat. No. 6,004,746 issued 21 Dec. 1999. Algorithms are also available inthe art for genome-based predictions of protein function (see, e.g.,Marcotte, et al., Nature 402: 4 Nov. 1999, 83-86).

Alternatively one can screen peptide libraries to identify moleculesthat interact with 205P1B5 protein sequences. In such methods, peptidesthat bind to 205P1B5 are identified by screening libraries that encode arandom or controlled collection of amino acids. Peptides encoded by thelibraries are expressed as fusion proteins of bacteriophage coatproteins, the bacteriophage particles are then screened against the205P1B5 protein.

Accordingly, peptides having a wide variety of uses, such astherapeutic, prognostic or diagnostic reagents, are thus identifiedwithout any prior information on the structure of the expected ligand orreceptor molecule. Typical peptide libraries and screening methods thatcan be used to identify molecules that interact with 205P1B5 proteinsequences are disclosed for example in U.S. Pat. No. 5,723,286 issued 3Mar. 1998 and U.S. Pat. No. 5,733,731 issued 31 Mar. 1998.

Alternatively, cell lines that express 205P1B5 are used to identifyprotein-protein interactions mediated by 205P1B5. Such interactions canbe examined using immunoprecipitation techniques (see, e.g., Hamilton BJ, et al. Biochem. Biophys. Res. Commun. 1999, 261 :646-51). 205P1B5protein can be immunoprecipitated from 205P1B5-expressing cell linesusing anti-205P1B5 antibodies. Alternatively, antibodies against His-tagcan be used in a cell line engineered to express fusions of 205P1B5 anda His-tag (vectors mentioned above). The immunoprecipitated complex canbe examined for protein association by procedures such as Westernblotting, ³⁵S-methionine labeling of proteins, protein microsequencing,silver staining and two-dimensional gel electrophoresis.

Small molecules and ligands that interact with 205P1B5 can be identifiedthrough related embodiments of such screening assays. For example, smallmolecules can be identified that interfere with protein function,including molecules that interfere with 205P1B5's ability to mediatephosphorylation and de-phosphorylation, interaction with DNA or RNAmolecules as an indication of regulation of cell cycles, secondmessenger signaling or tumorigenesis. Similarly, small molecules thatmodulate 205P1B5-related ion channel, protein pump, or cellcommunication functions are identified and used to treat patients thathave a cancer that expresses 205P1B5 (see, e.g., Hille, B., IonicChannels of Excitable Membranes 2^(nd) Ed., Sinauer Assoc., Sunderland,Mass., 1992). Moreover, ligands that regulate 205P1B5 function can beidentified based on their ability to bind 205P1B5 and activate areporter construct Typical methods are discussed for example in U.S.Pat. No. 5,928,868 issued 27 Jul. 1999, and include methods for forminghybrid ligands in which at least one ligand is a small molecule. In anillustrative embodiment, cells engineered to express a fusion protein of205P1B5 and a DNA-binding protein are used to co-express a fusionprotein of a hybrid ligand/small molecule and a cDNA librarytranscriptional activator protein. The cells further contain a reportergene, the expression of which is conditioned on the proximity of thefirst and second fusion proteins to each other, an event that occursonly if the hybrid ligand binds to target sites on both hybrid proteins.Those cells that express the reporter gene are selected and the unknownsmall molecule or the unknown ligand is identified. This method providesa means of identifying modulators which activate or inhibit 205P1B5.

An embodiment of this invention comprises a method of screening for amolecule that interacts with an 205P1B5 amino acid sequence shown inFIG. 2 or FIG. 3, comprising the steps of contacting a population ofmolecules with the 205P1B5 amino acid sequence, allowing the populationof molecules and the 205P1B5 amino acid sequence to interact underconditions that facilitate an interaction, determining the presence of amolecule that interacts with the 205P1B5 amino acid sequence, and thenseparating molecules that do not interact with the 205P1B5 amino acidsequence from molecules that do. In a specific embodiment, the methodfurther comprises purifying, characterizing and identifying a moleculethat interacts with the 205P1B5 amino acid sequence. The identifiedmolecule can be used to modulate a function performed by 205P1B5. In apreferred embodiment, the 205P1B5 amino acid sequence is contacted witha library of peptides.

X.) Therapeutic Methods and Compositions

The identification of 205P1B5 as a protein that is normally expressed ina restricted set of tissues, but which is also expressed in prostate andother cancers, opens a number of therapeutic approaches to the treatmentof such cancers. As contemplated herein, 205P1B5 functions as atranscription factor involved in activating tumor-promoting genes orrepressing genes that block tumorigenesis.

Accordingly, therapeutic approaches that inhibit the activity of the205P1B5 protein are useful for patients suffering from a cancer thatexpresses 205P1B5. These therapeutic approaches generally fall into twoclasses. One class comprises various methods for inhibiting the bindingor association of the 205P1B5 protein with its binding partner or withother proteins. Another class comprises a variety of methods forinhibiting the transcription of the 205P1B5 gene or translation of205P1B5 mRNA.

X.A.) Anti-Cancer Vaccines

The invention provides cancer vaccines comprising a 205P1B5 -relatedprotein or 205P1B5-related nucleic acid. In view of the expression of205P1B5, cancer vaccines prevent and/or treat 205P1B5-expressing cancerswith minimal or no effects on non-target tissues. The use of a tumorantigen in a vaccine that generates humoral and/or cell-mediated immuneresponses as anti-cancer therapy is well known in the art and has beenemployed in prostate cancer using human PSMA and rodent PAP immunogens(Hodge et al., 1995, Int. J. Cancer 63:231-237; Fong et al., 1997, J.Immunol. 159:3113-3117).

Such methods can be readily practiced by employing a 205P1B5-relatedprotein, or an 205P1B5-encoding nucleic acid molecule and recombinantvectors capable of expressing and presenting the 205P1B5 immunogen(which typically comprises a number of antibody or T cell epitopes).Skilled artisans understand that a wide variety of vaccine systems fordelivery of immunoreactive epitopes are known in the art (see, e.g.,Heryln et al., Ann Med 1999 Feb. 31(1):66-78; Maruyama et al., CancerImmunol Immunother 2000 June 49(3):123-32) Briefly, such methods ofgenerating an immune response (e.g. humoral and/or cell-mediated) in amammal, comprise the steps of: exposing the mammal's immune system to animmunoreactive epitope (e.g. an epitope present in the 205P1B5 proteinshown in SEQ ID NO: 703 or analog or homolog thereof) so that the mammalgenerates an immune response that is specific for that epitope (e.g.generates antibodies that specifically recognize that epitope). In apreferred method, the 205P1B5 immunogen contains a biological motif, seee.g., Tables V-XVIII, or a peptide of a size range from 205P1B5indicated in FIG. 5, FIG. 6, FIG. 7, FIG. 8, and FIG. 9.

The entire 205P1B5 protein, immunogenic regions or epitopes thereof canbe combined and delivered by various means. Such vaccine compositionscan include, for example, lipopeptides (e.g., Vitiello, A. et al., J.Clin. Invest. 95:341, 1995), peptide compositions encapsulated inpoly(DL-lactide-co-glycolide) (“PLG”) microspheres (see, e.g., Eldridge,et al., Molec. Immunol. 28:287-294, 1991: Alonso et al., Vaccine12:299-306, 1994; Jones et al., Vaccine 13:675-681, 1995), peptidecompositions contained in immune stimulating complexes (ISCOMS) (see,e.g., Takahashi et al., Nature 344:873-875, 1990; Hu et al., Clin ExpImmunol. 113:235-243, 1998), multiple antigen peptide systems (MAPs)(see e.g., Tam, J. P., Proc. Natl. Acad. Sci. U.S.A. 85:5409-5413, 1988;Tam, J. P., J. Immunol. Methods 196:17-32, 1996), peptides formulated asmultivalent peptides; peptides for use in ballistic delivery systems,typically crystallized peptides, viral delivery vectors (Perkus, M. E.et al., In: Concepts in vaccine development, Kaufmann, S. H. E., ed., p.379, 1996; Chakrabarti, S. et al., Nature 320:535, 1986; Hu, S. L. etal., Nature 320:537, 1986; Kieny, M.-P. et al., AIDS Bio/Technology4:790, 1986; Top, F. H. et al., J. Infect. Dis. 124:148, 1971; Chanda,P. K. et al., Virology 175:535, 1990), particles of viral or syntheticorigin (e.g., Kofler, N. et al., J. Immunol. Methods. 192:25, 1996;Eldridge, J. H. et al., Sem. Hematol. 30:16, 1993; Falo, L. D., Jr. etal., Nature Med. 7:649, 1995), adjuvants (Warren, H. S., Vogel, F. R.,and Chedid, L. A. Annu. Rev. Immunol. 4:369, 1986; Gupta, R. K. et al.,Vaccine 11:293, 1993), liposomes (Reddy, R. et al., J. Immunol.148:1585, 1992; Rock, K. L., Immunol. Today 17:131, 1996), or, naked orparticle absorbed cDNA (Ulmer, J. B. et al., Science 259:1745, 1993;Robinson, H. L., Hunt, L. A., and Webster, R. G., Vaccine 11:957, 1993;Shiver, J. W. et al., In: Concepts in vaccine development, Kaufmann, S.H. E., ed., p. 423, 1996; Cease, K. B., and Berzofsky, J. A., Annu. Rev.Immunol. 12:923, 1994 and Eldridge, J. H. et al., Sem. Hematol. 30:16,1993). Toxin-targeted delivery technologies, also known as receptormediated targeting, such as those of Avant Immunotherapeutics, Inc.(Needham, Mass.) may also be used.

In patients with 205P1B5-associated cancer, the vaccine compositions ofthe invention can also be used in conjunction with other treatments usedfor cancer, e.g., surgery, chemotherapy, drug therapies, radiationtherapies, etc. including use in combination with immune adjuvants suchas IL-2, IL-12, GM-CSF, and the like.

Cellular Vaccines:

CTL epitopes can be determined using specific algorithms to identifypeptides within 205P1B5 protein that bind corresponding HLA alleles (seee.g., Table IV; Epimer™ and Epimatrix™, Brown University (URL world wideweb URL brown.edu/Research/TB-HIV_Lab/epimatrix/epimatrix.html); and,BIMAS, (URL bimas.dcrt.nih.gov/; SYFPEITHI at URLsyfpeithi.bmi-heidelberg.com/). In a preferred embodiment, the 205P1B5immunogen contains one or more amino acid sequences identified usingtechniques well known in the art, such as the sequences shown in TablesV-XVIII or a peptide of 8, 9, 10 or 11 amino acids specified by an HLAClass I motif/supermotif (e.g., Table IV (A), Table IV (D), or Table IV(E)) and/or a peptide of at least 9 amino acids that comprises an HLAClass II motif/supermotif (e.g., Table IV (B) or Table IV (C)). As isappreciated in the art, the HLA Class I binding groove is essentiallyclosed ended so that peptides of only a particular size range can fitinto the groove and be bound, generally HLA Class I epitopes are 8, 9,10, or 11 amino acids long. In contrast, the HLA Class II binding grooveis essentially open ended; therefore a peptide of about 9 or more aminoacids can be bound by an HLA Class II molecule. Due to the bindinggroove differences between HLA Class I and II, HLA Class I motifs arelength specific, i.e., position two of a Class I motif is the secondamino acid in an amino to carboxyl direction of the peptide. The aminoacid positions in a Class II motif are relative only to each other, notthe overall peptide, i.e., additional amino acids can be attached to theamino and/or carboxyl termini of a motif-bearing sequence. HLA Class IIepitopes are often 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, or 25 amino acids long, or longer than 25 amino acids.

Antibody-Based Vaccines

A wide variety of methods for generating an immune response in a mammalare known in the art (for example as the first step in the generation ofhybridomas). Methods of generating an immune response in a mammalcomprise exposing the mammal's immune system to an immunogenic epitopeon a protein (e.g. the 205P1B5 protein) so that an immune response isgenerated. A typical embodiment consists of a method for generating animmune response to 205P1B5 in a host, by contacting the host with asufficient amount of at least one 205P1B5 B cell or cytotoxic T-cellepitope or analog thereof; and at least one periodic interval thereafterre-contacting the host with the 205P1B5 B cell or cytotoxic T-cellepitope or analog thereof. A specific embodiment consists of a method ofgenerating an immune response against a 205P1B5 -related protein or aman-made multiepitopic peptide comprising: administering 205P1B5immunogen (e.g. the 205P1B5 protein or a peptide fragment thereof, an205P1B5 fusion protein or analog etc.) in a vaccine preparation to ahuman or another mammal. Typically, such vaccine preparations furthercontain a suitable adjuvant (see, e.g., U.S. Pat. No. 6,146,635) or auniversal helper epitope such as a PADRE™ peptide (Epimmune Inc., SanDiego, Calif.; see, e.g., Alexander et al., J. Immunol. 2000 164(3);164(3): 1625-1633; Alexander et al., Immunity 1994 1(9): 751-761 andAlexander et al., Immunol. Res. 1998 18(2): 79-92). An alternativemethod comprises generating an immune response in an individual againsta 205P1B5 immunogen by: administering in vivo to muscle or skin of theindividual's body a DNA molecule that comprises a DNA sequence thatencodes an 205P1B5 immunogen, the DNA sequence operatively linked toregulatory sequences which control the expression of the DNA sequence;wherein the DNA molecule is taken up by cells, the DNA sequence isexpressed in the cells and an immune response is generated against theimmunogen (see, e.g., U.S. Pat. No. 5,962,428). Optionally a geneticvaccine facilitator such as anionic lipids; saponins; lectins;estrogenic compounds; hydroxylated lower alkyls; dimethyl sulfoxide; andurea is also administered.

Nucleic Acid Vaccines:

Vaccine compositions of the invention include nucleic acid-mediatedmodalities. DNA or RNA that encode protein(s) of the invention can beadministered to a patient Genetic immunization methods can be employedto generate prophylactic or therapeutic humoral and cellular immuneresponses directed against cancer cells expressing 205P1B5. Constructscomprising DNA encoding a 205P1B5-related protein/immunogen andappropriate regulatory sequences can be injected directly into muscle orskin of an individual, such that the cells of the muscle or skin take-upthe construct and express the encoded 205P1B5 protein/immunogen.Alternatively, a vaccine comprises a 205P1B5 -related protein.Expression of the 205P1B5-related protein immunogen results in thegeneration of prophylactic or therapeutic humoral and cellular immunityagainst cells that bear 205P1B5 protein. Various prophylactic andtherapeutic genetic immunization techniques known in the art can be used(for review, see information and references published at Internetaddress world wide web URL genweb.com). Nucleic acid-based delivery isdescribed, for instance, in Wolff et. al., Science 247:1465 (1990) aswell as U.S. Pat. Nos. 5,580,859; 5,589,466; 5,804,566; 5,739,118;5,736,524; 5,679,647; WO 98/04720. Examples of DNA-based deliverytechnologies include “naked DNA”, facilitated (bupivicaine, polymers,peptide-mediated) delivery, cationic lipid complexes, andparticle-mediated (“gene gun”) or pressure-mediated delivery (see, e.g.,U.S. Pat. No. 5,922,687).

For therapeutic or prophylactic immunization purposes, proteins of theinvention can be expressed via viral or bacterial vectors. Various viralgene delivery systems that can be used in the practice of the inventioninclude, but are not limited to, vaccinia, fowlpox, canarypox,adenovirus, influenza, poliovirus, adeno-associated virus, lentivirus,and sindbis virus (see, e.g., Restifo, 1996, Curr. Opin. Immunol.8:658-663; Tsang et al. J. Natl. Cancer Inst. 87:982-990 (1995)).Non-viral delivery systems can also be employed by introducing naked DNAencoding a 205P1B5-related protein into the patient (e.g.,intramuscularly or intradermally) to induce an anti-tumor response.

Vaccinia virus is used, for example, as a vector to express nucleotidesequences that encode the peptides of the invention. Upon introductioninto a host, the recombinant vaccinia virus expresses the proteinimmunogenic peptide, and thereby elicits a host immune response.Vaccinia vectors and methods useful in immunization protocols aredescribed in, e.g., U.S. Pat. No. 4,722,848. Another vector is BCG(Bacille Calmette Guerin). BCG vectors are described in Stover et al.,Nature 351:456-460(1991). A wide variety of other vectors useful fortherapeutic administration or immunization of the peptides of theinvention, e.g. adeno and adeno-associated virus vectors, retroviralvectors, Salmonella typhi vectors, detoxified anthrax toxin vectors, andthe like, will be apparent to those skilled in the art from thedescription herein.

Thus, gene delivery systems are used to deliver a 205P1B5 -relatednucleic acid molecule. In one embodiment, the full-length human 205P1B5cDNA is employed. In another embodiment 205P1B5 nucleic acid moleculesencoding specific cytotoxic T lymphocyte (CTL) and/or antibody epitopesare employed.

Ex Vivo Vaccines

Various ex vivo strategies can also be employed to generate an immuneresponse. One approach involves the use of antigen presenting cells(APCs) such as dendritic cells (DC) to present 205P1B5 antigen to apatient's immune system. Dendritic cells express MHC class I and IImolecules, B7 co-stimulator, and IL-12, and are thus highly specializedantigen presenting cells. In prostate cancer, autologous dendritic cellspulsed with peptides of the prostate-specific membrane antigen (PSMA)are being used in a Phase I clinical trial to stimulate prostate cancerpatients' immune systems (Tjoa et al., 1996, Prostate 28:65-69; Murphyet al., 1996, Prostate 29:371-380). Thus, dendritic cells can be used topresent 205P1B5 peptides to T cells in the context of MHC class I or IImolecules. In one embodiment, autologous dendritic cells are pulsed with205P1B5 peptides capable of binding to MHC class I and/or class IImolecules. In another embodiment, dendritic cells are pulsed with thecomplete 205P1B5 protein. Yet another embodiment involves engineeringthe overexpression of the 205P1B5 gene in dendritic cells using variousimplementing vectors known in the art, such as adenovirus (Arthur etal., 1997, Cancer Gene Ther. 4:17-25), retrovirus (Henderson et al.,1996, Cancer Res. 56:3763-3770), lentivirus, adeno-associated virus, DNAtransfection (Ribas et al., 1997, Cancer Res. 57:2865-2869), ortumor-derived RNA transfection (Ashley et al., 1997, J. Exp. Med.186:1177-1182). Cells that express 205P1B5 can also be engineered toexpress immune modulators, such as GM-CSF, and used as immunizingagents.

X.)B.) 205P1B5 as a Target for Antibody-Based Therapy

205P1B5 is an attractive target for antibody-based therapeuticstrategies. A number of antibody strategies are known in the art fortargeting both extracellular and intracellular molecules (see, e.g.,complement and ADCC mediated killing as well as the use of intrabodies).Because 205P1B5 is expressed by cancer cells of various lineagesrelative to corresponding normal cells, systemic administration of205P1B5 -immunoreactive compositions are prepared that exhibit excellentsensitivity without toxic, non-specific and/or non-target effects causedby binding of the immunoreactive composition to non-target organs andtissues. Antibodies specifically reactive with domains of 205P1B5 areuseful to treat 205P1B5 -expressing cancers systemically, either asconjugates with a toxin or therapeutic agent, or as naked antibodiescapable of inhibiting cell proliferation or function.

205P1B5 antibodies can be introduced into a patient such that theantibody binds to 205P1B5 and modulates a function, such as aninteraction with a binding partner, and consequently mediatesdestruction of the tumor cells and/or inhibits the growth of the tumorcells. Mechanisms by which such antibodies exert a therapeutic effectcan include complement-mediated cytolysis, antibody-dependent cellularcytotoxicity, modulation of the physiological function of 205P1B5,inhibition of ligand binding or signal transduction pathways, modulationof tumor cell differentiation, alteration of tumor angiogenesis factorprofiles, and/or apoptosis.

Those skilled in the art understand that antibodies can be used tospecifically target and bind immunogenic molecules such as animmunogenic region of the 205P1B5 sequence shown in FIG. 2 or FIG. 3. Inaddition, skilled artisans understand that it is routine to conjugateantibodies to cytotoxic agents (see, e.g., Slevers et al. Blood 93:113678-3684 (Jun. 1, 1999)). When cytotoxic and/or therapeutic agents aredelivered directly to cells, such as by conjugating them to antibodiesspecific for a molecule expressed by that cell (e.g. 205P1B5), thecytotoxic agent will exert its known biological effect (i.e.cytotoxicity) on those cells.

A wide variety of compositions and methods for using antibody-cytotoxicagent conjugates to kill cells are known in the art. In the context ofcancers, typical methods entail administering to an animal having atumor a biologically effective amount of a conjugate comprising aselected cytotoxic and/or therapeutic agent linked to a targeting agent(e.g. an anti-205P1B5 antibody) that binds to a marker (e.g. 205P1B5)expressed, accessible to binding or localized on the cell surfaces. Atypical embodiment is a method of delivering a cytotoxic and/ortherapeutic agent to a cell expressing 205P1B5, comprising conjugatingthe cytotoxic agent to an antibody that immunospecifically binds to a205P1B5 epitope, and, exposing the cell to the antibody-agent conjugate.Another illustrative embodiment is a method of treating an individualsuspected of suffering from metastasized cancer, comprising a step ofadministering parenterally to said individual a pharmaceuticalcomposition comprising a therapeutically effective amount of an antibodyconjugated to a cytotoxic and/or therapeutic agent.

Cancer immunotherapy using anti-205P1B5 antibodies can be done inaccordance with various approaches that have been successfully employedin the treatment of other types of cancer, including but not limited tocolon cancer (Arlen et al., 1998, Crit. Rev. Immunol. 18:133-138),multiple myeloma (Ozaki et al., 1997, Blood 90:3179-3186, Tsunenari etal., 1997, Blood 90:2437-2444), gastric cancer (Kasprzyk et al., 1992,Cancer Res. 52:2771-2776), B-cell lymphoma (Funakoshi et al., 1996, J.Immunother. Emphasis Tumor Immunol. 19:93-101), leukemia (Zhong et al.,1996, Leuk. Res. 20:581-589), colorectal cancer (Moun et al., 1994,Cancer Res. 54:6160-6166; Velders et al., 1995, Cancer Res.55:4398-4403), and breast cancer (Shepard et al., 1991, J. Clin.Immunol. 11:117-127). Some therapeutic approaches involve conjugation ofnaked antibody to a toxin, such as the conjugation of Y⁹¹ or I¹³¹ toanti-CD20 antibodies (e.g., Zevalin™, IDEC Pharmaceuticals Corp. orBexxar™, Coulter Pharmaceuticals), while others involveco-administration of antibodies and other therapeutic agents, such asHerceptin™ (trastuzumab) with paclitaxel (Genentech, Inc.). To treatprostate cancer, for example, 205P1B5 antibodies can be administered inconjunction with radiation, chemotherapy or hormone ablation.

Although 205P1B5 antibody therapy is useful for all stages of cancer,antibody therapy can be particularly appropriate in advanced ormetastatic cancers. Treatment with the antibody therapy of the inventionis indicated for patients who have received one or more rounds ofchemotherapy. Alternatively, antibody therapy of the invention iscombined with a chemotherapeutic or radiation regimen for patients whohave not received chemotherapeutic treatment. Additionally, antibodytherapy can enable the use of reduced dosages of concomitantchemotherapy, particularly for patients who do not tolerate the toxicityof the chemotherapeutic agent very well.

Cancer patients can be evaluated for the presence and level of 205P1B5expression, preferably using immunohistochemical assessments of tumortissue, quantitative 205P1B5 imaging, or other techniques that reliablyindicate the presence and degree of 205P1B5 expression.Immunohistochemical analysis of tumor biopsies or surgical specimens ispreferred for this purpose. Methods for immunohistochemical analysis oftumor tissues are well known in the art.

Anti-205P1B5 monoclonal antibodies that treat prostate and other cancersinclude those that initiate a potent immune response against the tumoror those that are directly cytotoxic. In this regard, anti-205P1B5monoclonal antibodies (mAbs) can elicit tumor cell lysis by eithercomplement-mediated or antibody-dependent cell cytotoxicity (ADCC)mechanisms, both of which require an intact Fc portion of theimmunoglobulin molecule for interaction with effector cell Fc receptorsites on complement proteins. In addition, anti-205P1B5 mAbs that exerta direct biological effect on tumor growth are useful to treat cancersthat express 205P1B5. Mechanisms by which directly cytotoxic mAbs actinclude: inhibition of cell growth, modulation of cellulardifferentiation, modulation of tumor angiogenesis factor profiles, andthe induction of apoptosis. The mechanism(s) by which a particularanti-205P1B5 mAb exerts an anti-tumor effect is evaluated using anynumber of in vitro assays that evaluate cell death such as ADCC, ADMMC,complement-mediated cell lysis, and so forth, as is generally known inthe art.

In some patients, the use of murine or other non-human monoclonalantibodies, or human/mouse chimeric mAbs can induce moderate to strongimmune responses against the non-human antibody. This can result inclearance of the antibody from circulation and reduced efficacy. In themost severe cases, such an immune response can lead to the extensiveformation of immune complexes which, potentially, can cause renalfailure. Accordingly, preferred monoclonal antibodies used in thetherapeutic methods of the invention are those that are either fullyhuman or humanized and that bind specifically to the target 205P1B5antigen with high affinity but exhibit low or no antigenicity in thepatient.

Therapeutic methods of the invention contemplate the administration ofsingle anti-205P1B5 mAbs as well as combinations, or cocktails, ofdifferent mAbs. Such mAb cocktails can have certain advantages inasmuchas they contain mAbs that target different epitopes, exploit differenteffector mechanisms or combine directly cytotoxic mAbs with mAbs thatrely on immune effector functionality. Such mAbs in combination canexhibit synergistic therapeutic effects. In addition, anti-205P1B5 mAbscan be administered concomitantly with other therapeutic modalities,including but not limited to various chemotherapeutic agents,androgen-blockers, immune modulators (e.g., IL-2, GM-CSF), surgery orradiation. The anti-205P1B5 mAbs are administered in their “naked” orunconjugated form, or can have a therapeutic agent(s) conjugated tothem.

Anti-205P1B5 antibody formulations are administered via any routecapable of delivering the antibodies to a tumor cell. Routes ofadministration include, but are not limited to, intravenous,intraperitoneal, intramuscular, intratumor, intradermal, and the like.Treatment generally involves repeated administration of the anti-205P1B5antibody preparation, via an acceptable route of administration such asintravenous injection (IV), typically at a dose in the range of about0.1 to about 10 mg/kg body weight In general, doses in the range of10-500 mg mAb per week are effective and well tolerated.

Based on clinical experience with the Herceptin mAb in the treatment ofmetastatic breast cancer, an initial loading dose of approximately 4mg/kg patient body weight IV, followed by weekly doses of about 2 mg/kgIV of the anti-205P1B5 mAb preparation represents an acceptable dosingregimen. Preferably, the initial loading dose is administered as a 90minute or longer infusion. The periodic maintenance dose is administeredas a 30 minute or longer infusion, provided the initial dose was welltolerated. As appreciated by those of skill in the art, various factorscan influence the ideal dose regimen in a particular case. Such factorsinclude, for example, the binding affinity and half life of the Ab ormAbs used, the degree of 205P1B5 expression in the patient, the extentof circulating shed 205P1B5 antigen, the desired steady-state antibodyconcentration level, frequency of treatment, and the influence ofchemotherapeutic or other agents used in combination with the treatmentmethod of the invention, as well as the health status of a particularpatient.

Optionally, patients should be evaluated for the levels of 205P1B5 in agiven sample (e.g. the levels of circulating 205P1B5 antigen and/or205P1B5 expressing cells) in order to assist in the determination of themost effective dosing regimen, etc. Such evaluations are also used formonitoring purposes throughout therapy, and are useful to gaugetherapeutic success in combination with the evaluation of otherparameters (for example, urine cytology and/or ImmunoCyt levels inbladder cancer therapy, or by analogy, serum PSA levels in prostatecancer therapy).

Anti-idiotypic anti-205P1B5 antibodies can also be used in anti-cancertherapy as a vaccine for inducing an immune response to cells expressinga 205P1B5-related protein. In particular, the generation ofanti-idiotypic antibodies is well known in the art; this methodology canreadily be adapted to generate anti-idiotypic anti-205P1B5 antibodiesthat mimic an epitope on a 205P1B5 -related protein (see, for example,Wagner et al., 1997, Hybridoma 16:33-40; Foon et al., 1995, J. Clin.Invest. 96:334-342; Herlyn et al., 1996, Cancer Immunol. Immunother.43:65-76). Such an anti-idiotypic antibody can be used in cancer vaccinestrategies.

X.C.) 205P1B5 as a Target for Cellular Immune Responses

Vaccines and methods of preparing vaccines that contain animmunogenically effective amount of one or more HLA-binding peptides asdescribed herein are further embodiments of the invention. Furthermore,vaccines in accordance with the invention encompass compositions of oneor more of the claimed peptides. A peptide can be present in a vaccineindividually. Alternatively, the peptide can exist as a homopolymercomprising multiple copies of the same peptide, or as a heteropolymer ofvarious peptides. Polymers have the advantage of increased immunologicalreaction and, where different peptide epitopes are used to make up thepolymer, the additional ability to induce antibodies and/or CTLs thatreact with different antigenic determinants of the pathogenic organismor tumor-related peptide targeted for an immune response. Thecomposition can be a naturally occurring region of an antigen or can beprepared, e.g., recombinantly or by chemical synthesis.

Carriers that can be used with vaccines of the invention are well knownin the art, and include, e.g., thyroglobulin, albumins such as humanserum albumin, tetanus toxoid, polyamino acids such as poly L-lysine,poly L-glutamic acid, influenza, hepatitis B virus core protein, and thelike. The vaccines can contain a physiologically tolerable (i.e.,acceptable) diluent such as water, or saline, preferably phosphatebuffered saline. The vaccines also typically include an adjuvant.Adjuvants such as incomplete Freund's adjuvant, aluminum phosphate,aluminum hydroxide, or alum are examples of materials well known in theart. Additionally, as disclosed herein, CTL responses can be primed byconjugating peptides of the invention to lipids, such astripalmitoyl-S-glycerylcysteinlyseryl-serine (P₃CSS). Moreover, anadjuvant such as a syntheticcytosine-phosphorothiolated-guanine-containing (CpG) oligonucleotideshas been found to increase CTL responses 10- to 100-fold. (see, e.g.Davila and Celis J. Immunol. 165:539-547 (2000))

Upon immunization with a peptide composition in accordance with theinvention, via injection, aerosol oral, transdermal, transmucosal,intrapleural, intrathecal, or other suitable routes, the immune systemof the host responds to the vaccine by producing large amounts of CTLsand/or HTLs specific for the desired antigen. Consequently, the hostbecomes at least partially immune to later development of cells thatexpress or overexpress 205P1B5 antigen, or derives at least sometherapeutic benefit when the antigen was tumor-associated.

In some embodiments, it may be desirable to combine the class I peptidecomponents with components that induce or facilitate neutralizingantibody and or helper T cell responses directed to the target antigen.A preferred embodiment of such a composition comprises class I and classII epitopes in accordance with the invention. An alternative embodimentof such a composition comprises a class I and/or class II epitope inaccordance with the invention, along with a cross reactive HTL epitopesuch as PADRE™ (Epimmune, San Diego, Calif.) molecule (described e.g.,in U.S. Pat. No. 5,736,142).

A vaccine of the invention can also include antigen-presenting cells(APC), such as dendritic cells (DC), as a vehicle to present peptides ofthe invention. Vaccine compositions can be created in vitro, followingdendritic cell mobilization and harvesting, whereby loading of dendriticcells occurs in vitro. For example, dendritic cells are transfected,e.g., with a minigene in accordance with the invention, or are pulsedwith peptides. The dendritic cell can then be administered to a patientto elicit immune responses in vivo. Vaccine compositions, either DNA- orpeptide-based, can also be administered in vivo in combination withdendritic cell mobilization whereby loading of dendritic cells occurs invivo.

Preferably, the following principles are utilized when selecting anarray of epitopes for inclusion in a polyepitopic composition for use ina vaccine, or for selecting discrete epitopes to be included in avaccine and/or to be encoded by nucleic acids such as a minigene. It ispreferred that each of the following principles be balanced in order tomake the selection. The multiple epitopes to be incorporated in a givenvaccine composition may be, but need not be, contiguous in sequence inthe native antigen from which the epitopes are derived.

1.) Epitopes are selected which, upon administration, mimic immuneresponses that have been observed to be correlated with tumor clearance.For HLA Class I this includes 3-4 epitopes that come from at least onetumor associated antigen (TAA). For HLA Class II a similar rationale isemployed; again 34 epitopes are selected from at least one TAA (see,e.g., Rosenberg et al., Science 278:1447-1450). Epitopes from one TAAmay be used in combination with epitopes from one or more additionalTAAs to produce a vaccine that targets tumors with varying expressionpatterns of frequently-expressed TAAs.

2.) Epitopes are selected that have the requisite binding affinityestablished to be correlated with immunogenicity: for HLA Class I anIC₅₀ of 500 nM or less, often 200 nM or less; and for Class II an IC₅₀of 1000 nM or less.

3.) Sufficient supermotif bearing-peptides, or a sufficient array ofallele-specific motif-bearing peptides, are selected to give broadpopulation coverage. For example, it is preferable to have at least 80%population coverage. A Monte Carlo analysis, a statistical evaluationknown in the art, can be employed to assess the breadth, or redundancyof population coverage.

4.) When selecting epitopes from cancer-related antigens it is oftenuseful to select analogs because the patient may have developedtolerance to the native epitope.

5.) Of particular relevance are epitopes referred to as “nestedepitopes.” Nested epitopes occur where at least two epitopes overlap ina given peptide sequence. A nested peptide sequence can comprise B cell,HLA class I and/or HLA class II epitopes. When providing nestedepitopes, a general objective is to provide the greatest number ofepitopes per sequence. Thus, an aspect is to avoid providing a peptidethat is any longer than the amino terminus of the amino terminal epitopeand the carboxyl terminus of the carboxyl terminal epitope in thepeptide. When providing a multi-epitopic sequence, such as a sequencecomprising nested epitopes, it is generally important to screen thesequence in order to insure that it does not have pathological or otherdeleterious biological properties.

6.) If a polyepitopic protein is created, or when creating a minigene,an objective is to generate the smallest peptide that encompasses theepitopes of interest. This principle is similar, if not the same as thatemployed when selecting a peptide comprising nested epitopes. However,with an artificial polyepitopic peptide, the size minimization objectiveis balanced against the need to integrate any spacer sequences betweenepitopes in the polyepitopic protein. Spacer amino acid residues can,for example, be introduced to avoid junctional epitopes (an epitoperecognized by the immune system, not present in the target antigen, andonly created by the man-made juxtaposition of epitopes), or tofacilitate cleavage between epitopes and thereby enhance epitopepresentation. Junctional epitopes are generally to be avoided becausethe recipient may generate an immune response to that non-nativeepitope. Of particular concern is a junctional epitope that is a“dominant epitope.” A dominant epitope may lead to such a zealousresponse that immune responses to other epitopes are diminished orsuppressed.

7.) Where the sequences of multiple variants of the same target proteinare present, potential peptide epitopes can also be selected on thebasis of their conservancy. For example, a criterion for conservancy maydefine that the entire sequence of an HLA class I binding peptide or theentire 9-mer core of a class II binding peptide be conserved in adesignated percentage of the sequences evaluated for a specific proteinantigen.

X.C.1. Minigene Vaccines

A number of different approaches are available which allow simultaneousdelivery of multiple epitopes. Nucleic acids encoding the peptides ofthe invention are a particularly useful embodiment of the invention.Epitopes for inclusion in a minigene are preferably selected accordingto the guidelines set forth in the previous section. A preferred meansof administering nucleic acids encoding the peptides of the inventionuses minigene constructs encoding a peptide comprising one or multipleepitopes of the invention.

The use of multi-epitope minigenes is described below and in, Ishioka etal., J. Immunol. 162:3915-3925, 1999; An, L. and Whitton, J. L., J.Virol. 71:2292, 1997; Thomson, S. A. et al., J. Immunol. 157:822, 1996;Whitton, J. L. et al., J. Virol. 67:348, 1993; Hanke, R. et al., Vaccine16:426, 1998. For example, a multi-epitope DNA plasmid encodingsupermotif- and/or motif-bearing epitopes derived 205P1B5, the PADRE®universal helper T cell epitope (or multiple HTL epitopes from 205P1B5),and an endoplasmic reticulum-translocating signal sequence can beengineered. A vaccine may also comprise epitopes that are derived fromother TAAs.

The immunogenicity of a multi-epitopic minigene can be confirmed intransgenic mice to evaluate the magnitude of CTL induction responsesagainst the epitopes tested. Further, the immunogenicity of DNA-encodedepitopes in vivo can be correlated with the in vitro responses ofspecific CTL lines against target cells transfected with the DNAplasmid. Thus, these experiments can show that the minigene serves toboth: 1.) generate a CTL response and 2.) that the induced CTLsrecognized cells expressing the encoded epitopes.

For example, to create a DNA sequence encoding the selected epitopes(minigene) for expression in human cells, the amino acid sequences ofthe epitopes may be reverse translated. A human codon usage table can beused to guide the codon choice for each amino acid. Theseepitope-encoding DNA sequences may be directly adjoined, so that whentranslated, a continuous polypeptide sequence is created. To optimizeexpression and/or immunogenicity, additional elements can beincorporated into the minigene design. Examples of amino acid sequencesthat can be reverse translated and included in the minigene sequenceinclude: HLA class I epitopes, HLA class II epitopes, antibody epitopes,a ubiquitination signal sequence, and/or an endoplasmic reticulumtargeting signal. In addition, HLA presentation of CTL and HTL epitopesmay be improved by including synthetic (e.g. poly alanine) ornaturally-occurring flanking sequences adjacent to the CTL or HTLepitopes; these larger peptides comprising the epitope(s) are within thescope of the invention.

The minigene sequence may be converted to DNA by assemblingoligonucleotides that encode the plus and minus strands of the minigene.Overlapping oligonucleotides (30-100 bases long) may be synthesized,phosphorylated, purified and annealed under appropriate conditions usingwell known techniques. The ends of the oligonucleotides can be joined,for example, using T4 DNA ligase. This synthetic minigene, encoding theepitope polypeptide, can then be cloned into a desired expressionvector.

Standard regulatory sequences well known to those of skill in the artare preferably included in the vector to ensure expression in the targetcells. Several vector elements are desirable: a promoter with adown-stream cloning site for minigene insertion; a polyadenylationsignal for efficient transcription termination; an E. coli origin ofreplication; and an E. coli selectable marker (e.g. ampicillin orkanamycin resistance). Numerous promoters can be used for this purpose,e.g., the human cytomegalovirus (hCMV) promoter. See, e.g., U.S. Pat.Nos. 5,580,859 and 5,589,466 for other suitable promoter sequences.

Additional vector modifications may be desired to optimize minigeneexpression and immunogenicity. In some cases, introns are required forefficient gene expression, and one or more synthetic ornaturally-occurring introns could be incorporated into the transcribedregion of the minigene. The inclusion of mRNA stabilization sequencesand sequences for replication in mammalian cells may also be consideredfor increasing minigene expression.

Once an expression vector is selected, the minigene is cloned into thepolylinker region downstream of the promoter. This plasmid istransformed into an appropriate E. coli strain, and DNA is preparedusing standard techniques. The orientation and DNA sequence of theminigene, as well as all other elements included in the vector, areconfirmed using restriction mapping and DNA sequence analysis. Bacterialcells harboring the correct plasmid can be stored as a master cell bankand a working cell bank.

In addition, immunostimulatory sequences (ISSs or CpGs) appear to play arole in the immunogenicity of DNA vaccines. These sequences may beincluded in the vector, outside the minigene coding sequence, if desiredto enhance immunogenicity.

In some embodiments, a bi-cistronic expression vector which allowsproduction of both the minigene-encoded epitopes and a second protein(included to enhance or decrease immunogenicity) can be used. Examplesof proteins or polypeptides that could beneficially enhance the immuneresponse if co-expressed include cytokines (e.g., IL-2, IL-12, GM-CSF),cytokine-inducing molecules (e.g., LeIF), costimulatory molecules, orfor HTL responses, pan-DR binding proteins (PADRE™, Epimmune, San Diego,Calif.). Helper (HTL) epitopes can be joined to intracellular targetingsignals and expressed separately from expressed CTL epitopes; thisallows direction of the HTL epitopes to a cell compartment differentthan that of the CTL epitopes. If required, this could facilitate moreefficient entry of HTL epitopes into the HLA class II pathway, therebyimproving HTL induction. In contrast to HTL or CTL induction,specifically decreasing the immune response by co-expression ofimmunosuppressive molecules (e.g. TGF-β) may be beneficial in certaindiseases.

Therapeutic quantities of plasmid DNA can be produced for example, byfermentation in E. coli, followed by purification. Aliquots from theworking cell bank are used to inoculate growth medium and grown tosaturation in shaker flasks or a bioreactor according to well-knowntechniques. Plasmid DNA can be purified using standard bioseparationtechnologies such as solid phase anion-exchange resins supplied byQIAGEN, Inc. (Valencia, Calif.). If required, supercoiled DNA can beisolated from the open circular and linear forms using gelelectrophoresis or other methods.

Purified plasmid DNA can be prepared for injection using a variety offormulations. The simplest of these is reconstitution of lyophilized DNAin sterile phosphate-buffer saline (PBS). This approach, known as “nakedDNA,” is currently being used for intramuscular (IM) administration inclinical trials. To maximize the immunotherapeutic effects of minigeneDNA vaccines, an alternative method for formulating purified plasmid DNAmay be desirable. A variety of methods have been described, and newtechniques may become available. Cationic lipids, glycolipids, andfusogenic liposomes can also be used in the formulation (see, e.g., asdescribed. by WO 93/24640; Mannino & Gould-Fogerite, BioTechniques 6(7):682(1988); U.S. Pat. No. 5,279,833; WO 91/06309; and Felgner, et al.,Proc. Nat'l Acad. Sci. USA 84:7413 (1987). In addition, peptides andcompounds referred to collectively as protective, interactive,non-condensing compounds (PINC) could also be complexed to purifiedplasmid DNA to influence variables such as stability, intramusculardispersion, or trafficking to specific organs or cell types.

Target cell sensitization can be used as a functional assay forexpression and HLA class I presentation of minigene-encoded CTLepitopes. For example, the plasmid DNA is introduced into a mammaliancell line that is suitable as a target for standard CTL chromium releaseassays. The transfection method used will be dependent on the finalformulation. Electroporation can be used for “naked” DNA, whereascationic lipids allow direct in vitro transfection. A plasmid expressinggreen fluorescent protein (GFP) can be co-transfected to allowenrichment of transfected cells using fluorescence activated cellsorting (FACS). These cells are then chromium-51 (⁵¹Cr) labeled and usedas target cells for epitope-specific CTL lines; cytolysis, detected by⁵¹Cr release, indicates both production of, and HLA presentation of,minigene-encoded CTL epitopes. Expression of HTL epitopes may beevaluated in an analogous manner using assays to assess HTL activity.

In vivo immunogenicity is a second approach for functional testing ofminigene DNA formulations. Transgenic mice expressing appropriate humanHLA proteins are immunized with the DNA product. The dose and route ofadministration are formulation dependent (e.g., IM for DNA in PBS,intraperitoneal (i.p.) for lipid-complexed DNA). Twenty-one days afterimmunization, splenocytes are harvested and restimulated for one week inthe presence of peptides encoding each epitope being tested. Thereafter,for CTL effector cells, assays are conducted for cytolysis ofpeptide-loaded, ⁵¹Cr-labeled target cells using standard techniques.Lysis of target cells that were sensitized by HLA loaded with peptideepitopes, corresponding to minigene-encoded epitopes, demonstrates DNAvaccine function for in vivo induction of CTLs. Immunogenicity of HTLepitopes is confirmed in transgenic mice in an analogous manner.

Alternatively, the nucleic acids can be administered using ballisticdelivery as described, for instance, in U.S. Pat. No. 5,204,253. Usingthis technique, particles comprised solely of DNA are administered. In afurther alternative embodiment, DNA can be adhered to particles, such asgold particles.

Minigenes can also be delivered using other bacterial or viral deliverysystems well known in the art, e.g., an expression construct encodingepitopes of the invention can be incorporated into a viral vector suchas vaccinia.

X.C.2. Combinations of CTL Peptides with Helper Peptides

Vaccine compositions comprising CTL peptides of the invention can bemodified, e.g., analoged, to provide desired attributes, such asimproved serum half life, broadened population coverage or enhancedimmunogenicity.

For instance, the ability of a peptide to induce CTL activity can beenhanced by linking the peptide to a sequence which contains at leastone epitope that is capable of inducing a T helper cell response.Although a CTL peptide can be directly linked to a T helper peptide,often CTL epitope/HTL epitope conjugates are linked by a spacermolecule. The spacer is typically comprised of relatively small, neutralmolecules, such as amino acids or amino acid mimetics, which aresubstantially uncharged under physiological conditions. The spacers aretypically selected from, e.g., Ala, Gly, or other neutral spacers ofnonpolar amino acids or neutral polar amino acids. It will be understoodthat the optionally present spacer need not be comprised of the sameresidues and thus may be a hetero- or homo-oligomer. When present, thespacer will usually be at least one or two residues, more usually threeto six residues and sometimes 10 or more residues. The CTL peptideepitope can be linked to the T helper peptide epitope either directly orvia a spacer either at the amino or carboxy terminus of the CTL peptide.The amino terminus of either the immunogenic peptide or the T helperpeptide may be acylated.

In certain embodiments, the T helper peptide is one that is recognizedby T helper cells present in a majority of a genetically diversepopulation. This can be accomplished by selecting peptides that bind tomany, most, or all of the HLA class II molecules. Examples of such aminoacid bind many HLA Class II molecules include sequences from antigenssuch as tetanus toxoid at positions 830-843 (QYIKANSKFIGITE; SEQ ID NO:710), Plasmodium falciparum circumsporozoite (CS) protein at positions378-398 (DIEKKIAKMEKASSVFNVVNS; SEQ ID NO: 711), and Streptococcus 18 kDprotein at positions 116-131 (GAVDSILGGVATYGAA; SEQ ID NO: 712). Otherexamples include peptides bearing a DR 1-4-7 supermotif, or either ofthe DR3 motifs.

Alternatively, it is possible to prepare synthetic peptides capable ofstimulating T helper lymphocytes, in a loosely HLA-restricted fashion,using amino acid sequences not found in nature (see, e.g., PCTpublication WO 95/07707). These synthetic compounds calledPan-DR-binding epitopes (e.g., PADRE™, Epimmune, Inc., San Diego,Calif.) are designed to most preferably bind most HLA-DR (human HLAclass II) molecules. For instance, a pan-DR-binding epitope peptidehaving the formula: aKXVAAWTLKAAa (SEQ ID NO: 713), where “X” is eithercyclohexylalanine, phenylalanine, or tyrosine, and a is either D-alanineor L-alanine, has been found to bind to most HLA-DR alleles, and tostimulate the response of T helper lymphocytes from most individuals,regardless of their HLA type. An alternative of a pan-DR binding epitopecomprises all “L” natural amino acids and can be provided in the form ofnucleic acids that encode the epitope.

HTL peptide epitopes can also be modified to alter their biologicalproperties. For example, they can be modified to include D-amino acidsto increase their resistance to proteases and thus extend their serumhalf life, or they can be conjugated to other molecules such as lipids,proteins, carbohydrates, and the like to increase their biologicalactivity. For example, a T helper peptide can be conjugated to one ormore palmitic acid chains at either the amino or carboxyl termini.

X.C.3. Combinations of CTL Peptides with T Cell Priming Agents

In some embodiments it may be desirable to include in the pharmaceuticalcompositions of the invention at least one component which primes Blymphocytes or T lymphocytes. Lipids have been identified as agentscapable of priming CTL in vivo. For example, palmitic acid residues canbe attached to the ε-and α-amino groups of a lysine residue and thenlinked, e.g., via one or more linking residues such as Gly, Gly-Gly-,Ser, Ser-Ser, or the like, to an immunogenic peptide. The lipidatedpeptide can then be administered either directly in a micelle orparticle, incorporated into a liposome, or emulsified in an adjuvant,e.g., incomplete Freund's adjuvant. In a preferred embodiment, aparticularly effective immunogenic composition comprises palmitic acidattached to ε- and α-amino groups of Lys, which is attached via linkage,e.g., Ser-Ser, to the amino terminus of the immunogenic peptide.

As another example of lipid priming of CTL responses, E. colilipoproteins, such as tripalmitoyl-S-glycerylcysteinlyseryl-serine(P₃CSS) can be used to prime virus specific CTL when covalently attachedto an appropriate peptide (see, e.g., Deres, et al., Nature 342:561,1989). Peptides of the invention can be coupled to P₃CSS, for example,and the lipopeptide administered to an individual to specifically primean immune response to the target antigen. Moreover, because theinduction of neutralizing antibodies can also be primed withP₃CSS-conjugated epitopes, two such compositions can be combined to moreeffectively elicit both humoral and cell-mediated responses.

X.C.4. Vaccine Compositions Comprising DC Pulsed with CTL and/or HTLPeptides

An embodiment of a vaccine composition in accordance with the inventioncomprises ex vivo administration of a cocktail of epitope-bearingpeptides to PBMC, or isolated DC therefrom, from the patient's blood. Apharmaceutical to facilitate harvesting of DC can be used, such asProgenipoietin™ (Pharmacia-Monsanto, St. Louis, Mo.) or GM-CSF/IL-4.After pulsing the DC with peptides and prior to reinfusion intopatients, the DC are washed to remove unbound peptides. In thisembodiment, a vaccine comprises peptide-pulsed DCs which present thepulsed peptide epitopes complexed with HLA molecules on their surfaces.

The DC can be pulsed ex vivo with a cocktail of peptides, some of whichstimulate CTL responses to 205P1B5. Optionally, a helper T cell (HTL)peptide, such as a natural or artificial loosely restricted HLA Class IIpeptide, can be included to facilitate the CTL response. Thus, a vaccinein accordance with the invention is used to treat a cancer whichexpresses or overexpresses 205P1B5.

X.D. Adoptive Immunotherapy

Antigenic 205P1B5 -related peptides are used to elicit a CTL and/or HTLresponse ex vivo, as well. The resulting CTL or HTL cells, can be usedto treat tumors in patients that do not respond to other conventionalforms of therapy, or will not respond to a therapeutic vaccine peptideor nucleic acid in accordance with the invention. Ex vivo CTL or HTLresponses to a particular antigen are induced by incubating in tissueculture the patient's, or genetically compatible, CTL or HTL precursorcells together with a source of antigen-presenting cells (APC), such asdendritic cells, and the appropriate immunogenic peptide. After anappropriate incubation time (typically about 7-28 days), in which theprecursor cells are activated and expanded into effector cells, thecells are infused back into the patient, where they will destroy (CTL)or facilitate destruction (HTL) of their specific target cell (e.g., atumor cell). Transfected dendritic cells may also be used as antigenpresenting cells.

X.E. Administration of Vaccines for Therapeutic or Prophylactic Purposes

Pharmaceutical and vaccine compositions of the invention are typicallyused to treat and/or prevent a cancer that expresses or overexpresses205P1B5. In therapeutic applications, peptide and/or nucleic acidcompositions are administered to a patient in an amount sufficient toelicit an effective B cell, CTL and/or HTL response to the antigen andto cure or at least partially arrest or slow symptoms and/orcomplications. An amount adequate to accomplish this is defined as“therapeutically effective dose.” Amounts effective for this use willdepend on, e.g., the particular composition administered, the manner ofadministration, the stage and severity of the disease being treated, theweight and general state of health of the patient, and the judgment ofthe prescribing physician.

For pharmaceutical compositions, the immunogenic peptides of theinvention, or DNA encoding them, are generally administered to anindividual already bearing a tumor that expresses 205P1B5. The peptidesor DNA encoding them can be administered individually or as fusions ofone or more peptide sequences. Patients can be treated with theimmunogenic peptides separately or in conjunction with other treatments,such as surgery, as appropriate.

For therapeutic use, administration should generally begin at the firstdiagnosis of 205P1B5 -associated cancer. This is followed by boostingdoses until at least symptoms are substantially abated and for a periodthereafter. The embodiment of the vaccine composition (i.e., including,but not limited to embodiments such as peptide cocktails, polyepitopicpolypeptides, minigenes, or TAA-specific CTLs or pulsed dendritic cells)delivered to the patient may vary according to the stage of the diseaseor the patient's health status. For example, in a patient with a tumorthat expresses 205P1B5, a vaccine comprising 205P1B5-specific CTL may bemore efficacious in killing tumor cells in patient with advanced diseasethan alternative embodiments.

It is generally important to provide an amount of the peptide epitopedelivered by a mode of administration sufficient to effectivelystimulate a cytotoxic T cell response; compositions which stimulatehelper T cell responses can also be given in accordance with thisembodiment of the invention.

The dosage for an initial therapeutic immunization generally occurs in aunit dosage range where the lower value is about 1, 5, 50, 500, or 1,000μg and the higher value is about 10,000; 20,000; 30,000; or 50,000 μg.Dosage values for a human typically range from about 500 μg to about50,000 μg per 70 kilogram patient. Boosting dosages of between about 1.0μg to about 50,000 μg of peptide pursuant to a boosting regimen overweeks to months may be administered depending upon the patients responseand condition as determined by measuring the specific activity of CTLand HTL obtained from the patient's blood. Administration shouldcontinue until at least clinical symptoms or laboratory tests indicatethat the neoplasia, has been eliminated or reduced and for a periodthereafter. The dosages, routes of administration, and dose schedulesare adjusted in accordance with methodologies known in the art.

In certain embodiments, the peptides and compositions of the presentinvention are employed in serious disease states, that is,life-threatening or potentially life threatening situations. In suchcases, as a result of the minimal amounts of extraneous substances andthe relative nontoxic nature of the peptides in preferred compositionsof the invention, it is possible and may be felt desirable by thetreating physician to administer substantial excesses of these peptidecompositions relative to these stated dosage amounts.

The vaccine compositions of the invention can also be used purely asprophylactic agents. Generally the dosage for an initial prophylacticimmunization generally occurs in a unit dosage range where the lowervalue is about 1, 5, 50, 500, or 1000 μg and the higher value is about10,000; 20,000; 30,000; or 50,000 μg. Dosage values for a humantypically range from about 500 μg to about 50,000 μg per 70 kilogrampatient. This is followed by boosting dosages of between about 1.0 μg toabout 50,000 μg of peptide administered at defined intervals from aboutfour weeks to six months after the initial administration of vaccine.The immunogenicity of the vaccine can be assessed by measuring thespecific activity of CTL and HTL obtained from a sample of the patient'sblood.

The pharmaceutical compositions for therapeutic treatment are intendedfor parenteral, topical, oral, nasal, intrathecal, or local (e.g. as acream or topical ointment) administration. Preferably, thepharmaceutical compositions are administered parentally, e.g.,intravenously, subcutaneously, intradermally, or intramuscularly. Thus,the invention provides compositions for parenteral administration whichcomprise a solution of the immunogenic peptides dissolved or suspendedin an acceptable carrier, preferably an aqueous carrier.

A variety of aqueous carriers may be used, e.g., water, buffered water,0.8% saline, 0.3% glycine, hyaluronic acid and the like. Thesecompositions may be sterilized by conventional, well-known sterilizationtechniques, or may be sterile filtered. The resulting aqueous solutionsmay be packaged for use as is, or lyophilized, the lyophilizedpreparation being combined with a sterile solution prior toadministration.

The compositions may contain pharmaceutically acceptable auxiliarysubstances as required to approximate physiological conditions, such aspH-adjusting and buffering agents, tonicity adjusting agents, wettingagents, preservatives, and the like, for example, sodium acetate, sodiumlactate, sodium chloride, potassium chloride, calcium chloride, sorbitanmonolaurate, triethanolamine oleate, etc.

The concentration of peptides of the invention in the pharmaceuticalformulations can vary widely, i.e., from less than about 0.1%, usuallyat or at least about 2% to as much as 20% to 50% or more by weight, andwill be selected primarily by fluid volumes, viscosities, etc., inaccordance with the particular mode of administration selected.

A human unit dose form of a composition is typically included in apharmaceutical composition that comprises a human unit dose of anacceptable carrier, in one embodiment an aqueous carrier, and isadministered in a volume/quantity that is known by those of skill in theart to be used for administration of such compositions to humans (see,e.g., Remington's Pharmaceutical Sciences, 17^(th) Edition, A. Gennaro,Editor, Mack Publishing Co., Easton, Pa., 1985). For example a peptidedose for initial immunization can be from about 1 to about 50,000 μg,generally 100-5,000 μg, for a 70 kg patient. For example, for nucleicacids an initial immunization may be performed using an expressionvector in the form of naked nucleic acid administered IM (or SC or ID)in the amounts of 0.5-5 mg at multiple sites. The nucleic acid (0.1 to1000 μg) can also be administered using a gene gun. Following anincubation period of 3-4 weeks, a booster dose is then administered. Thebooster can be recombinant fowlpox virus administered at a dose of 5−10⁷to 5×10⁹ pfu. For antibodies, a treatment generally involves repeatedadministration of the anti-205P1B5 antibody preparation, via anacceptable route of administration such as intravenous injection (IV),typically at a dose in the range of about 0.1 to about 10 mg/kg bodyweight In general, doses in the range of 10-500 mg mAb per week areeffective and well tolerated. Moreover, an initial loading dose ofapproximately 4 mg/kg patient body weight IV, followed by weekly dosesof about 2 mg/kg IV of the anti-205P1B5 mAb preparation represents anacceptable dosing regimen. As appreciated by those of skill in the art,various factors can influence the ideal dose in a particular case. Suchfactors include, for example, half life of a composition, the bindingaffinity of an Ab, the immunogenicity of a substance, the degree of205P1B5 expression in the patient, the extent of circulating shed205P1B5 antigen, the desired steady-state concentration level, frequencyof treatment, and the influence of chemotherapeutic or other agents usedin combination with the treatment method of the invention, as well asthe health status of a particular patient.

In one embodiment, human unit dose forms of polynucleotides comprise asuitable dosage range or effective amount that provides any therapeuticeffect. As appreciated by one of ordinary skill in the art a therapeuticeffect depends on a number of factors, including the sequence of thepolynucleotide, molecular weight of the polynucleotide and route ofadministration. Dosages are generally selected by the physician or otherhealth care professional in accordance with a variety of parametersknown in the art, such as severity of symptoms, history of the patientand the like. Generally, for a polynucleotide of about 20 bases, adosage range may be selected from, for example, an independentlyselected lower limit such as about 0.1, 0.25, 0.5, 1, 2, 5, 10, 20, 30,40, 50, 60, 80, 100, 200, 300, 400 or 500 mg/kg up to an independentlyselected upper limit greater than the lower limit, of about 60, 80, 100,200, 300, 400, 500, 750, 1000, 1500, 2000, 3000, 4000, 5000, 6000, 7000,8000, 9000 or 10,000 mg/kg. For example, a dose may be about any of thefollowing: 0.1 to 100 mg/kg, 0.1 to 50 mg/kg, 0.1 to 25 mg/kg, 0.1 to 10mg/kg, 1 to 500 mg/kg, 100 to 400 mg/kg, 200 to 300 mg/kg, 1 to 100mg/kg, 100 to 200 mg/kg, 300 to 400 mg/kg, 400 to 500 mg/kg, 500 to 1000mg/kg, 500 to 5000 mg/kg, or 500 to 10,000 mg/kg. Generally, parenteralroutes of administration may require higher doses of polynucleotidecompared to more direct application to the nucleotide to diseasedtissue, as do polynucleotides of increasing length.

In one embodiment, human unit dose forms of T-cells comprise a suitabledosage range or effective amount that provides any therapeutic effect.As appreciated by one of ordinary skill in the art, a therapeutic effectdepends on a number of factors. Dosages are generally selected by thephysician or other health care professional in accordance with a varietyof parameters known in the art, such as severity of symptoms, history ofthe patient and the like. A dose may be about 10⁴ cells to about 10⁶cells, about 10⁶ cells to about 10⁸ cells, about 10⁸ to about 10¹¹cells, or about 10⁸ to about 5×10¹⁰ cells. A dose may also about 10⁶cells/m² to about 10¹⁰ cells/m², or about 10⁶ cells/m² to about 10⁸cells/m².

Proteins(s) of the invention, and/or nucleic acids encoding theprotein(s), can also be administered via liposomes, which may also serveto: 1) target the proteins(s) to a particular tissue, such as lymphoidtissue; 2) to target selectively to diseases cells; or, 3) to increasethe half-life of the peptide composition. Liposomes include emulsions,foams, micelles, insoluble monolayers, liquid crystals, phospholipiddispersions, lamellar layers and the like. In these preparations, thepeptide to be delivered is incorporated as part of a liposome, alone orin conjunction with a molecule which binds to a receptor prevalent amonglymphoid cells, such as monoclonal antibodies which bind to the CD45antigen, or with other therapeutic or immunogenic compositions. Thus,liposomes either filled or decorated with a desired peptide of theinvention can be directed to the site of lymphoid cells, where theliposomes then deliver the peptide compositions. Liposomes for use inaccordance with the invention are formed from standard vesicle-forminglipids, which generally include neutral and negatively chargedphospholipids and a sterol, such as cholesterol. The selection of lipidsis generally guided by consideration of, e.g., liposome size, acidlability and stability of the liposomes in the blood stream. A varietyof methods are available for preparing liposomes, as described in, e.g.,Szoka, et al., Ann. Rev. Biophys. Bioeng. 9:467 (1980), and U.S. Pat.Nos. 4,235,871, 4,501,728, 4,837,028, and 5,019,369.

For targeting cells of the immune system, a ligand to be incorporatedinto the liposome can include, e.g., antibodies or fragments thereofspecific for cell surface determinants of the desired immune systemcells. A liposome suspension containing a peptide may be administeredintravenously, locally, topically, etc. in a dose which varies accordingto, inter alia, the manner of administration, the peptide beingdelivered, and the stage of the disease being treated.

For solid compositions, conventional nontoxic solid carriers may be usedwhich include, for example, pharmaceutical grades of mannitol, lactose,starch, magnesium stearate, sodium saccharin, talcum, cellulose,glucose, sucrose, magnesium carbonate, and the like. For oraladministration, a pharmaceutically acceptable nontoxic composition isformed by incorporating any of the normally employed excipients, such asthose carriers previously listed, and generally 10-95% of activeingredient, that is, one or more peptides of the invention, and morepreferably at a concentration of 25%-75%.

For aerosol administration, immunogenic peptides are preferably suppliedin finely divided form along with a surfactant and propellant. Typicalpercentages of peptides are about 0.01%-20% by weight, preferably about1%o-10%. The surfactant must, of course, be nontoxic, and preferablysoluble in the propellant. Representative of such agents are the estersor partial esters of fatty acids containing from about 6 to 22 carbonatoms, such as caproic, octanoic, lauric, palmitic, stearic, linoleic,linolenic, olesteric and oleic acids with an aliphatic polyhydricalcohol or its cyclic anhydride. Mixed esters, such as mixed or naturalglycerides may be employed. The surfactant may constitute about 0.1%-20%by weight of the composition, preferably about 0.25-5%. The balance ofthe composition is ordinarily propellant. A carrier can also beincluded, as desired, as with, e.g., lecithin for intranasal delivery.

XI.) Diagnostic and Prognostic Embodiments of 205P1B5.

As disclosed herein, 205P1B5 polynucleotides, polypeptides, reactivecytotoxic T cells (CTL), reactive helper T cells (HTL) andanti-polypeptide antibodies are used in well known diagnostic,prognostic and therapeutic assays that examine conditions associatedwith dysregulated cell growth such as cancer, in particular the cancerslisted in Table I (see, e.g., both its specific pattern of tissueexpression as well as its overexpression in certain cancers as describedfor example in Example 4).

205P1B5 can be analogized to a prostate associated antigen PSA, thearchetypal marker that has been used by medical practitioners for yearsto identify and monitor the presence of prostate cancer (see, e.g.,Merrill et al., J. Urol. 163(2): 503-5120 (2000); Polascik et al., J.Urol. August; 162(2):293-306 (1999) and Fortier et al., J. Nat. CancerInst. 91(19): 1635-1640(1999)). A variety of other diagnostic markersare also used in similar contexts including p53 and K-ras (see, e.g.,Tulchinsky et al., Int J Mol Med 1999 Jul. 4(1):99-102 and Minimoto etal., Cancer Detect Prev 2000;24(1):1-12). Therefore, this disclosure ofthe 205P1B5 polynucleotides and polypeptides (as well as the 205P1B5polynucleotide probes and anti-205P1B5 antibodies used to identify thepresence of these molecules) and their properties allows skilledartisans to utilize these molecules in methods that are analogous tothose used, for example, in a variety of diagnostic assays directed toexamining conditions associated with cancer.

Typical embodiments of diagnostic methods which utilize the 205P1B5polynucleotides, polypeptides, reactive T cells and antibodies areanalogous to those methods from well-established diagnostic assays whichemploy, e.g., PSA polynucleotides, polypeptides, reactive T cells andantibodies. For example, just as PSA polynucleotides are used as probes(for example in Northern analysis, see, e.g., Sharief et al., Biochem.Mol. Biol. Int. 33(3):567-74(1994)) and primers (for example in PCRanalysis, see, e.g., Okegawa et al., J. Urol. 163(4): 1189-1190 (2000))to observe the presence and/or the level of PSA mRNAs in methods ofmonitoring PSA overexpression or the metastasis of prostate cancers, the205P1B5 polynucleotides described herein can be utilized in the same wayto detect 205P1B5 overexpression or the metastasis of prostate and othercancers expressing this gene. Alternatively, just as PSA polypeptidesare used to generate antibodies specific for PSA which can then be usedto observe the presence and/or the level of PSA proteins in methods tomonitor PSA protein overexpression (see, e.g., Stephan et al., Urology55(4):560-3 (2000)) or the metastasis of prostate cells (see, e.g.,Alanen et al., Pathol. Res. Pract. 192(3):233-7 (1996)), the 205P1B5polypeptides described herein can be utilized to generate antibodies foruse in detecting 205P1B5 overexpression or the metastasis of prostatecells and cells of other cancers expressing this gene.

Specifically, because metastases involves the movement of cancer cellsfrom an organ of origin (such as the lung or prostate gland etc.) to adifferent area of the body (such as a lymph node), assays which examinea biological sample for the presence of cells expressing 205P1B5polynucleotides and/or polypeptides can be used to provide evidence ofmetastasis. For example, when a biological sample from tissue that doesnot normally contain 205P1B5 -expressing cells (lymph node) is found tocontain 205P1B5 -expressing cells such as the 205P1B5 expression seen inLAPC4 and LAPC9, xenografts isolated from lymph node and bonemetastasis, respectively, this finding is indicative of metastasis.

Alternatively 205P1B5 polynucleotides and/or polypeptides can be used toprovide evidence of cancer, for example, when cells in a biologicalsample that do not normally express 205P1B5 or express 205P1B5 at adifferent level are found to express 205P1B5 or have an increasedexpression of 205P1B5 (see, e.g., the 205P1B5 expression in the cancerslisted in Table I and in patient samples etc. shown in the accompanyingFigures). In such assays, artisans may further wish to generatesupplementary evidence of metastasis by testing the biological samplefor the presence of a second tissue restricted marker (in addition to205P1B5) such as PSA, PSCA etc. (see, e.g., Alanen et al., Pathol. Res.Pract. 192(3): 233-237 (1996)).

Just as PSA polynucleotide fragments and polynucleotide variants areemployed by skilled artisans for use in methods of monitoring PSA,205P1B5 polynucleotide fragments and polynucleotide variants are used inan analogous manner. In particular, typical PSA polynucleotides used inmethods of monitoring PSA are probes or primers which consist offragments of the PSA cDNA sequence. Illustrating this, primers used toPCR amplify a PSA polynucleotide must include less than the whole PSAsequence to function in the polymerase chain reaction. In the context ofsuch PCR reactions, skilled artisans generally create a variety ofdifferent polynucleotide fragments that can be used as primers in orderto amplify different portions of a polynucleotide of interest or tooptimize amplification reactions (see, e.g., Caetano-Anolles, G.Biotechniques 25(3): 472-476,478-480 (1998); Robertson et al., MethodsMol. Biol. 98:121-154 (1998)). An additional illustration of the use ofsuch fragments is provided in Example 4, where a 205P1B5 polynucleotidefragment is used as a probe to show the expression of 205P1B5 RNAs incancer cells. In addition, variant polynucleotide sequences aretypically used as primers and probes for the corresponding mRNAs in PCRand Northern analyses (see, e.g., Sawai et al., Fetal Diagn. Ther. 1996November-December 11(6):407-13 and Current Protocols In MolecularBiology, Volume 2, Unit 2, Frederick M. Ausubel et al. eds., 1995)).Polynucleotide fragments and variants are useful in this context wherethey are capable of binding to a target polynucleotide sequence (e.g.the 205P1B5 polynucleotide shown in SEQ ID NO: 701) under conditions ofhigh stringency.

Furthermore, PSA polypeptides which contain an epitope that can berecognized by an antibody or T cell that specifically binds to thatepitope are used in methods of monitoring PSA. 205P1B5 polypeptidefragments and polypeptide analogs or variants can also be used in ananalogous manner. This practice of using polypeptide fragments orpolypeptide variants to generate antibodies (such as anti-PSA antibodiesor T cells) is typical in the art with a wide variety of systems such asfusion proteins being used by practitioners (see, e.g., CurrentProtocols In Molecular Biology, Volume 2, Unit 16, Frederick M. Ausubelet al. eds., 1995). In this context, each epitope(s) functions toprovide the architecture with which an antibody or T cell is reactive.Typically, skilled artisans create a variety of different polypeptidefragments that can be used in order to generate immune responsesspecific for different portions of a polypeptide of interest (see, e.g.,U.S. Pat. No. 5,840,501 and U.S. Pat. No. 5,939,533). For example it maybe preferable to utilize a polypeptide comprising one of the 205P1B5biological motifs discussed herein or a motif-bearing subsequence whichis readily identified by one of skill in the art based on motifsavailable in the art. Polypeptide fragments, variants or analogs aretypically useful in this context as long as they comprise an epitopecapable of generating an antibody or T cell specific for a targetpolypeptide sequence (e.g. the 205P1B5 polypeptide shown in SEQ ID NO:703).

As shown herein, the 205P1B5 polynucleotides and polypeptides (as wellas the 205P1B5 polynucleotide probes and anti-205P1B5 antibodies or Tcells used to identify the presence of these molecules) exhibit specificproperties that make them useful in diagnosing cancers such as thoselisted in Table I. Diagnostic assays that measure the presence of205P1B5 gene products, in order to evaluate the presence or onset of adisease condition described herein, such as prostate cancer, are used toidentify patients for preventive measures or further monitoring, as hasbeen done so successfully with PSA Moreover, these materials satisfy aneed in the art for molecules having similar or complementarycharacteristics to PSA in situations where, for example, a definitediagnosis of metastasis of prostatic origin cannot be made on the basisof a test for PSA alone (see, e.g., Alanen et al., Pathol. Res. Pract.192(3): 233-237 (1996)), and consequently, materials such as 205P1B5polynucleotides and polypeptides (as well as the 205P1B5 polynucleotideprobes and anti-205P1B5 antibodies used to identify the presence ofthese molecules) must be employed to confirm metastases of prostaticorigin.

Finally, in addition to their use in diagnostic assays, the 205P1B5polynucleotides disclosed herein have a number of other utilities suchas their use in the identification of oncogenetic associated chromosomalabnormalities in the chromosomal region to which the 205P1B5 gene maps(see Example 3 below). Moreover, in addition to their use in diagnosticassays, the 205P1B5 -related proteins and polynucleotides disclosedherein have other utilities such as their use in the forensic analysisof tissues of unknown origin (see, e.g., Takahama K Forensic Sci Int1996 Jun. 28;80(1-2): 63-9).

Additionally, 205P1B5-related proteins or polynucleotides of theinvention can be used to treat a pathologic condition characterized bythe over-expression of 205P1B5. For example, the amino acid or nucleicacid sequence of FIG. 2 or FIG. 3, or fragments of either, can be usedto generate an immune response to the 205P1B5 antigen. Antibodies orother molecules that react with 205P1B5 can be used to modulate thefunction of this molecule, and thereby provide a therapeutic benefit.

XII.) Inhibition of 205P1B5 Protein Function

The invention includes various methods and compositions for inhibitingthe binding of 205P1B5 to its binding partner or its association withother protein(s) as well as methods for inhibiting 205P1B5 function.

XII.A.) Inhibition of 205P1B5 With Intracellular Antibodies

In one approach, a recombinant vector that encodes single chainantibodies that specifically bind to 205P1B5 are introduced into 205P1B5expressing cells via gene transfer technologies. Accordingly, theencoded single chain anti-205P1B5 antibody is expressed intracellularly,binds to 205P1B5 protein, and thereby inhibits its function. Methods forengineering such intracellular single chain antibodies are well known.Such intracellular antibodies, also known as “intrabodies”, arespecifically targeted to a particular compartment within the cell,providing control over where the inhibitory activity of the treatment isfocused. This technology has been successfully applied in the art (forreview, see Richardson and Marasco, 1995, TIBTECH vol. 13). Intrabodieshave been shown to virtually eliminate the expression of otherwiseabundant cell surface receptors (see, e.g., Richardson et al., 1995,Proc. Natl. Acad. Sci. USA 92: 3137-3141; Beerli et al., 1994, J. Biol.Chem. 289:23931-23936; Deshane et al., 1994, Gene Ther. 1: 332-337).

Single chain antibodies comprise the variable domains of the heavy andlight chain joined by a flexible linker polypeptide, and are expressedas a single polypeptide. Optionally, single chain antibodies areexpressed as a single chain variable region fragment joined to the lightchain constant region. Well-known intracellular trafficking signals areengineered into recombinant polynucleotide vectors encoding such singlechain antibodies in order to precisely target the intrabody to thedesired intracellular compartment. For example, intrabodies targeted tothe endoplasmic reticulum (ER) are engineered to incorporate a leaderpeptide and, optionally, a C-terminal ER retention signal, such as theKDEL amino acid motif Intrabodies intended to exert activity in thenucleus are engineered to include a nuclear localization signal. Lipidmoieties are joined to intrabodies in order to tether the intrabody tothe cytosolic side of the plasma membrane. Intrabodies can also betargeted to exert function in the cytosol. For example, cytosolicintrabodies are used to sequester factors within the cytosol, therebypreventing them from being transported to their natural cellulardestination.

In one embodiment, intrabodies are used to capture 205P1B5 in thenucleus, thereby preventing its activity within the nucleus. Nucleartargeting signals are engineered into such 205P1B5 intrabodies in orderto achieve the desired targeting. Such 205P1B5 intrabodies are designedto bind specifically to a particular 205P1B5 domain. In anotherembodiment, cytosolic intrabodies that specifically bind to the 205P1B5protein are used to prevent 205P1B5 from gaining access to the nucleus,thereby preventing it from exerting any biological activity within thenucleus (e.g., preventing 205P1B5 from forming transcription complexeswith other factors).

In order to specifically direct the expression of such intrabodies toparticular cells, the transcription of the intrabody is placed under theregulatory control of an appropriate tumor-specific promoter and/orenhancer. In order to target intrabody expression specifically toprostate, for example, the PSA promoter and/or promoter/enhancer can beutilized (See, for example, U.S. Pat. No. 5,919,652 issued 6 Jul. 1999).

XII.B.) Inhibition of 205P1B5 with Recombinant Proteins

In another approach, recombinant molecules bind to 205P1B5 and therebyinhibit 205P1B5 function. For example, these recombinant moleculesprevent or inhibit 205P1B5 from accessing/binding to its bindingpartner(s) or associating with other protein(s). Such recombinantmolecules can, for example, contain the reactive part(s) of a 205P1B5specific antibody molecule. In a particular embodiment, the 205P1B5binding domain of a 205P1B5 binding partner is engineered into a dimericfusion protein, whereby the fusion protein comprises two 205P1B5 ligandbinding domains linked to the Fc portion of a human IgG, such as humanIgG1. Such IgG portion can contain, for example, the C_(H)2 and C_(H)3domains and the hinge region, but not the C_(H)1 domain Such dimericfusion proteins are administer in soluble form to patients sufferingfrom a cancer associated with the expression of 205P1B5, whereby thedimeric fusion protein specifically binds to 205P1B5 and blocks 205P1B5interaction with a binding partner. Such dimeric fusion proteins arefurther combined into multimeric proteins using known antibody linkingtechnologies.

XII.C.) Inhibition of 205P1B5 Transcription or Translation

The present invention also comprises various methods and compositionsfor inhibiting the transcription of the 205P1B5 gene. Similarly, theinvention also provides methods and compositions for inhibiting thetranslation of 205P1B5 mRNA into protein.

In one approach, a method of inhibiting the transcription of the 205P1B5gene comprises contacting the 205P1B5 gene with a 205P1B5 antisensepolynucleotide. In another approach, a method of inhibiting 205P1B5 mRNAtranslation comprises contacting the 205P1B5 mRNA with an antisensepolynucleotide. In another approach, a 205P1B5 specific ribozyme is usedto cleave the 205P1B5 message, thereby inhibiting translation. Suchantisense and ribozyme based methods can also be directed to theregulatory regions of the 205P1B5 gene, such as the 205P1B5 promoterand/or enhancer elements. Similarly, proteins capable of inhibiting a205P1B5 gene transcription factor are used to inhibit 205P1B5 mRNAtranscription. The various polynucleotides and compositions useful inthe aforementioned methods have been described above. The use ofantisense and ribozyme molecules to inhibit transcription andtranslation is well known in the art.

Other factors that inhibit the transcription of 205P1B5 by interferingwith 205P1B5 transcriptional activation are also useful to treat cancersexpressing 205P1B5. Similarly, factors that interfere with 205P1B5processing are useful to treat cancers that express 205P1B5. Cancertreatment methods utilizing such factors are also within the scope ofthe invention.

XII.D.) General Considerations for Therapeutic Strategies

Gene transfer and gene therapy technologies can be used to delivertherapeutic polynucleotide molecules to tumor cells synthesizing 205P1B5(i.e., antisense, ribozyme, polynucleotides encoding intrabodies andother 205P1B5 inhibitory molecules). A number of gene therapy approachesare known in the art. Recombinant vectors encoding 205P1B5 antisensepolynucleotides, ribozymes, factors capable of interfering with 205P1B5transcription, and so forth, can be delivered to target tumor cellsusing such gene therapy approaches.

The above therapeutic approaches can be combined with any one of a widevariety of surgical, chemotherapy or radiation therapy regimens. Thetherapeutic approaches of the invention can enable the use of reduceddosages of chemotherapy (or other therapies) and/or less frequentadministration, an advantage for all patients and particularly for thosethat do not tolerate the toxicity of the chemotherapeutic agent well.

The anti-tumor activity of a particular composition (e.g., antisense,ribozyme, intrabody), or a combination of such compositions, can beevaluated using various in vitro and in vivo assay systems. In vitroassays that evaluate therapeutic activity include cell growth assays,soft agar assays and other assays indicative of tumor promotingactivity, binding assays capable of determining the extent to which atherapeutic composition will inhibit the binding of 205P1B5 to a bindingpartner, etc.

In vivo, the effect of a 205P1B5 therapeutic composition can beevaluated in a suitable animal model. For example, xenogenic prostatecancer models can be used, wherein human prostate cancer explants orpassaged xenograft tissues are introduced into immune compromisedanimals, such as nude or SCID mice (Klein et al., 1997, Nature Medicine3: 402408). For example, PCT Patent Application WO98/16628, Sawyers etal., published Apr. 23, 1998, describes various xenograft models ofhuman prostate cancer capable of recapitulating the development ofprimary tumors, micrometastasis, and the formation of osteoblasticmetastases characteristic of late stage disease. Efficacy can bepredicted using assays that measure inhibition of tumor formation, tumorregression or metastasis, and the like. In vivo assays that evaluate thepromotion of apoptosis are useful in evaluating therapeuticcompositions. In one embodiment, xenografts from tumor bearing micetreated with the therapeutic composition can be examined for thepresence of apoptotic foci and compared to untreated controlxenograft-bearing mice. The extent to which apoptotic foci are found inthe tumors of the treated mice provides an indication of the therapeuticefficacy of the composition.

The therapeutic compositions used in the practice of the foregoingmethods can be formulated into pharmaceutical compositions comprising acarrier suitable for the desired delivery method. Suitable carriersinclude any material that when combined with the therapeutic compositionretains the anti-tumor function of the therapeutic composition and isgenerally non-reactive with the patient's immune system. Examplesinclude, but are not limited to, any of a number of standardpharmaceutical carriers such as sterile phosphate buffered salinesolutions, bacteriostatic water, and the like (see, generally,Remington's Pharmaceutical Sciences 16^(th) Edition, A. Osal., Ed.,1980).

Therapeutic formulations can be solubilized and administered via anyroute capable of delivering the therapeutic composition to the tumorsite. Potentially effective routes of administration include, but arenot limited to, intravenous, parenteral, intraperitoneal, intramuscular,intratumor, intradermal, intraorgan, orthotopic, and the like. Apreferred formulation for intravenous injection comprises thetherapeutic composition in a solution of preserved bacteriostatic water,sterile unpreserved water, and/or diluted in polyvinylchloride orpolyethylene bags containing 0.9% sterile Sodium Chloride for Injection,USP. Therapeutic protein preparations can be lyophilized and stored assterile powders, preferably under vacuum, and then reconstituted inbacteriostatic water (containing for example, benzyl alcoholpreservative) or in sterile water prior to injection.

Dosages and administration protocols for the treatment of cancers usingthe foregoing methods will vary with the method and the target cancer,and will generally depend on a number of other factors appreciated inthe art.

XIII.) Kits

For use in the diagnostic and therapeutic applications described herein,kits are also within the scope of the invention. Such kits can comprisea carrier, package or container that is compartmentalized to receive oneor more containers such as vials, tubes, and the like, each of thecontainer(s) comprising one of the separate elements to be used in themethod. For example, the container(s) can comprise a probe that is orcan be detectably labeled. Such probe can be an antibody orpolynucleotide specific for a 205P1B5-related protein or a 205P1B5 geneor message, respectively. Where the method utilizes nucleic acidhybridization to detect the target nucleic acid, the kit can also havecontainers containing nucleotide(s) for amplification of the targetnucleic acid sequence and/or a container comprising a reporter-means,such as a biotin-binding protein, such as avidin or streptavidin, boundto a reporter molecule, such as an enzymatic, florescent, orradioisotope label. The kit can include all or part of the amino acidsequence of FIG. 2 or FIG. 3 or analogs thereof, or a nucleic acidmolecules that encodes such amino acid sequences.

The kit of the invention will typically comprise the container describedabove and one or more other containers comprising materials desirablefrom a commercial and user standpoint, including buffers, diluents,filters, needles, syringes, and package inserts with instructions foruse.

A label can be present on the container to indicate that the compositionis used for a specific therapy or non-therapeutic application, and canalso indicate directions for either in vivo or in vitro use, such asthose described above. Directions and or other information can also beincluded on an insert which is included with the kit.

EXAMPLES

Various aspects of the invention are further described and illustratedby way of the several examples that follow, none of which are intendedto limit the scope of the invention.

Example 1 SSH-Generated Isolation of a cDNA Fragment of the 205P1B5 Gene

To isolate genes that are over-expressed in prostate cancer we used theSuppression Subtractive Hybridization (SSH) procedure using cDNA derivedfrom prostate cancer tissues. The 205P1B5 SSH cDNA sequence was derivedfrom a subtraction consisting of a prostate cancer pool (patients withGleason scores 6 and 7) minus a mix of cDNAs derived from nine normaltissues (stomach, skeletal muscle, lung, brain, liver, kidney, pancreas,small intestine, and heart). By RT-PCR, the 205P1B5 cDNA was identifiedas highly expressed in the prostate cancer tissue pool, prostate cancerxenograft pool (LAPC4-AD, LAPC4-AI, LAPC9-AD, LAPC9-AI), and in themetastasis cancer pool with no expression observed in the vital tissuepools consisting of normal liver, kidney, lung, stomach, pancreas, andcolon.

The 205P1B5 SSH cDNA sequence of 289 bp matched the Homo sapienscholinergic receptor, nicotinic, alpha polypeptide 2 (neuronal; CHRNA2)with 221/225 (98%) identities (FIG. 1). The fill length 205P1B5/CHRNA2cDNA and ORF is described in FIG. 2 with the protein sequence listed inFIG. 3.

Materials and Methods

RNA Isolation:

Tumor tissues were homogenized in Trizol reagent (Life Technologies,Gibco BRL) using 10 ml/g tissue or 10 ml/10⁸ cells to isolate total RNA.Poly A RNA was purified from total RNA using Qiagen's Oligotex mRNA Miniand Midi kits. Total and mRNA were quantified by spectrophotometricanalysis (O.D. 260/280 nm) and analyzed by gel electrophoresis.

Oligonucleotides:

The following HPLC purified oligonucleotides were used.

DPNCDN (cDNA synthesis primer): 5′TTTTGATCAAGCTT₃₀3′ (SEQ ID NO: 714)Adaptor 1: 5′CTAATACGACTCACTATAGGGCTCGAGCGGCCGCCCGGGCAG3′ (SEQ ID NO:715) 3′GGCCCGTCCTAG5′ (SEQ ID NO: 716) Adaptor 2:5′GTAATACGACTCACTATAGGGCAGCGTGGTCGCGGCCGAG3′ (SEQ ID NO: 717)3′CGGCTCCTAG5′ (SEQ ID NO: 708) PCR primer 1: 5′CTAATACGACTCACTATAGGGC3′(SEQ ID NO: 709) Nested primer (NP)1: 5′TCGAGCGGCCGCCCGGGCAGGA3′ (SEQ IDNO: 718) Nested primer (NP)2: 5′AGCGTGGTCGCGGCCGAGGA3′ (SEQ ID NO: 719)

Suppression Subtractive Hybridization:

Suppression Subtractive Hybridization (SSH) was used to identify cDNAscorresponding to genes that may be differentially expressed in prostatecancer. The SSH reaction utilized cDNA from prostate cancer patientswith Gleason scores of 6 and 7. The gene 205P1B5 was derived from aprostate cancer pool, Gleason 6, 7 minus nine normal tissues. The SSHDNA sequence (FIG. 1) was identified.

The cDNA derived from nine normal tissues (stomach, skeletal muscle,lung, brain, liver, kidney, pancreas, small intestine, and heart) wasused as the source of the “driver” cDNA, while the cDNA from a pool ofprostate cancer patients with Gleason scores 6 and 7 was used as thesource of the “tester” cDNA. Double stranded cDNAs corresponding totester and driver cDNAs were synthesized from 2 μg of poly(A)⁺ RNAisolated from the relevant tissue, as described above, using CLONTECH'sPCR-Select cDNA Subtraction Kit and 1 ng of oligonucleotide DPNCDN asprimer. First- and second-strand synthesis were carried out as describedin the Kit's user manual protocol (CLONTECH Protocol No. PT1117-1,Catalog No. K1804-1). The resulting cDNA was digested with Dpn II for 3hrs at 37° C. Digested cDNA was extracted with phenol/chloroform (1:1)and ethanol precipitated.

Tester cDNA was generated by diluting 1 μl of Dpn II digested cDNA fromthe relevant tissue source (see above) (400 ng) in 5 μl of water. Thediluted cDNA (2 μl, 160 ng) was then ligated to 2 μl of Adaptor 1 andAdaptor 2 (10 μM), in separate ligation reactions, in a total volume of10 μl at 16° C. overnight, using 400 u of T4 DNA ligase (CLONTECH).Ligation was terminated with 1 μl of 0.2 M EDTA and heating at 72° C.for 5 min.

The first hybridization was performed by adding 1.5 μl (600 ng) ofdriver cDNA to each of two tubes containing 1.5 μl (20 ng) Adaptor 1-and Adaptor 2-ligated tester cDNA. In a final volume of 4 μl, thesamples were overlaid with mineral oil, denatured in an MJ Researchthermal cycler at 98° C. for 1.5 minutes, and then were allowed tohybridize for 8 hrs at 68° C. The two hybridizations were then mixedtogether with an additional 1 μl of fresh denatured driver cDNA and wereallowed to hybridize overnight at 68° C. The second hybridization wasthen diluted in 200 μl of 20 mM Hepes, pH 8.3, 50 mM NaCl, 0.2 mM EDTA,heated at 70° C. for 7 min. and stored at −20° C.

PCR Amplification, Cloning and Sequencing of Gene Fragments Generatedfrom SSH:

To amplify gene fragments resulting from SSH reactions, two PCRamplifications were performed. In the primary PCR reaction 1 μl of thediluted final hybridization mix was added to 1 μl of PCR primer 1 (10μM), 0.5 μl dNTP mix (10 μM), 2.5 μl 10×reaction buffer (CLONTECH) and0.5 μl 50×Advantage cDNA polymerase Mix (CLONTECH) in a final volume of25 μl. PCR 1 was conducted using the following conditions: 75° C. for 5min., 94° C. for 25 sec., then 27 cycles of 94° C. for 10 sec, 66° C.for 30 sec, 72° C. for 1.5 min. Five separate primary PCR reactions wereperformed for each experiment. The products were pooled and diluted 1:10with water. For the secondary PCR reaction, 1 μl from the pooled anddiluted primary PCR reaction was added to the same reaction mix as usedfor PCR 1, except that primers NP1 and NP2 (10 μM) were used instead ofPCR primer 1. PCR 2 was performed using 10-12 cycles of 94° C. for 10sec, 68° C. for 30 sec, and 72° C. for 1.5 minutes. The PCR productswere analyzed using 2% agarose gel electrophoresis.

The PCR products were inserted into pCR2.1 using the T/A vector cloningkit (Invitrogen). Transformed E. coli were subjected to blue/white andampicillin selection. White colonies were picked and arrayed into 96well plates and were grown in liquid culture overnight. To identifyinserts, PCR amplification was performed on 1 ml of bacterial cultureusing the conditions of PCR1 and NP1 and NP2 as primers. PCR productswere analyzed using 2% agarose gel electrophoresis.

Bacterial clones were stored in 20% glycerol in a 96 well format.Plasmid DNA was prepared, sequenced, and subjected to nucleic acidhomology searches of the GenBank, dBest, and NCI-CGAP databases.

RT-PCR Expression Analysis:

First strand cDNAs can be generated from 1 μg of mRNA with oligo(dT)12-18 priming using the Gibco-BRL Superscript Preamplificationsystem. The manufacturer's protocol was used which included anincubation for 50 min at 42° C. with reverse transcriptase followed byRNAse H treatment at 37° C. for 20 min. After completing the reaction,the volume can be increased to 200 μl with water prior to normalization.First strand cDNAs from 16 different normal human tissues can beobtained from Clontech.

Normalization of the first strand cDNAs from multiple tissues wasperformed by using the primers 5′atatcgccgcgctcgtcgtcgacaa3′ (SEQ ID NO:706) and 5′agccacacgcagctcattgtagaagg 3′ (SEQ ID NO:707) to amplifyβ-actin. First strand cDNA (5 μl) were amplified in a total volume of 50μl containing 0.4 μM primers, 0.2 μM each dNTPs, 1×PCR buffer (Clontech,10 mM Tris-HCL, 1.5 mM MgCl₂, 50 mM KCl, pH8.3) and 1×Klentaq DNApolymerase (Clontech). Five μl of the PCR reaction can be removed at 18,20, and 22 cycles and used for agarose gel electrophoresis. PCR wasperformed using an MJ Research thermal cycler under the followingconditions: Initial denaturation can be at 94° C. for 15 sec, followedby a 18, 20, and 22 cycles of 94° C. for 15, 65° C. for 2 min, 72° C.for 5 sec. A final extension at 72° C. was carried out for 2 min. Afteragarose gel electrophoresis, the band intensities of the 283 b.p.β-actin bands from multiple tissues were compared by visual inspection.Dilution factors for the first strand cDNAs were calculated to result inequal β-actin band intensities in all tissues after 22 cycles of PCR.Three rounds of normalization can be required to achieve equal bandintensities in all tissues after 22 cycles of PCR.

To determine expression levels of the 205P1B5 gene, 5 μl of normalizedfirst strand cDNA were analyzed by PCR using 26, and 30 cycles ofamplification, Semi-quantitative expression analysis can be achieved bycomparing the PCR products at cycle numbers that give light bandintensities.

A typical RT-PCR expression analysis is shown in FIG. 10. RT-PCRexpression analysis was performed on first strand cDNAs generated usingpools of tissues from multiple samples. The cDNAs were shown to benormalized using beta-actin PCR.

Example 2 Full Length Cloning of 205P1B5

To isolate genes that are involved in prostate cancer, an experiment wasconducted using prostate cancer patients with Gleason scores 6 and 7.

The gene 205P1B5 was derived from a prostate cancer pool minus ninenormal tissues subtraction. The SSH DNA sequence (FIG. 1) was designated205P1B5. cDNA clone 205P1B5-clone 1 consisting of the CHRNA2 ORF wasidentified from normal prostate cDNA. A single base pair variation wasidentified at position 760 with an A instead of a G when compared to theCHRNA2 sequence.

Example 3 Chromosomal Localization

Chromosomal localization can implicate genes in disease pathogenesis.Several chromosome mapping approaches are available in the art,including fluorescent in situ hybridization (FISH), human /hamsterradiation hybrid (RH) panels (Walter et al., 1994; Nature Genetics 7:22;Research Genetics, Huntsville Ala.), human-rodent somatic cell hybridpanels, such as is available from the Cornell Institute (Camden, N.J.),and genomic viewers utilizing BLAST homologies to sequenced and mappedgenomic clones (NCBI, Bethesda, Md.).

205P1B5 maps to chromosome 8p21-8p12, using 205P1B5 sequence and theNCBI BLAST tool: (world wide web URLncbi.nlm.nih.gov/genome/seq/page.cgi?F=HsBlast.html&&ORG=Hs).

This region has been implicated in prostate cancer (Xu et al., Am J. HumGenet 2001 August; 69(2):341-350).

Example 4 Expression Analysis of 205P1B5 in Normal Tissues and PatientSpecimens

Analysis of 205P1B5 by RT-PCR is shown in FIG. 10. First strand cDNA wasprepared from vital pool 1 (VP: liver, lung and kidney), vital pool 2(VP2: pancreas, colon and stomach), prostate xenograft pool (LAPC-4AD,LAPC4AI, LAPC-9AD, LAPC-9AI), prostate cancer pool, and cancermetastasis pool. Normalization was performed by PCR using primers toactin and GAPDH. Semi-quantitative PCR, using primers to 205P1B5, wasperformed at 26 and 30 cycles of amplification. Results show expressionof 205P1B5 in prostate cancer pool prostate xenograft pool, cancermetastasis pool, but not in VP1 and VP2.

Extensive Northern blot analysis of 205P1B5 in 16 human normal tissuesdemonstrated that 205P1B5 expression is tissue-restricted (FIG. 11). Twomultiple tissue northern blots (Clontech) with 2 μg of mRNA/lane, wereprobed with 205P1B5 sequence. Size standards in kilobases (kb) areindicated on the side. An approximately 5 kb transcript was detected inprostate and brain but not in any other normal tissues. A larger 205P1B5transcript of approximately 7.5 kb was only detected in liver.

Expression of 205P1B5 was assayed on a pool of 3 tumors isolated fromprostate cancer patients (PCP) and on normal tissues FIG. 12). Northernblots with 10 μg of total RNA/lane were probed with 205P1B5 sequence.Size standards in kilobases (kb) are indicated on the side. 205P1B5expression was seen in the prostate cancer pool and the normal prostatebut not in normal bladder (NB), normal kidney (NK), normal colon (NC).Northern blot analysis on individual prostate cancer patient specimensand prostate cancer xenografts is shown in FIG. 13. RNA was extractedfrom prostate cancer xenografts (LAPC4AD, LAPC4AI, LAPC-9AD, LAPC-9AI),prostate cancer cell line PC3, normal prostate (N), prostate tumors (T)and normal adjacent tissue (Nat) derived from prostate cancer patients.Northern blot with 10 μg of total RNA/lane was probed with 205P1B5sequence. Size standards in kilobases (kb) are indicated on the side.Results show expression of 205P1B5 in all prostate tumor specimenstested. Expression is also seen in 3 of the 4 xenografts, but not in thePC3 cell line.

The restricted expression of 205P1B5 in normal tissues and theexpression detected in the cancers listed in Table I indicate that205P1B5 is a therapeutic and prophylactic target and a diagnostic andprognostic marker for human cancer.

FIG. 15 shows expression of 205P1B5 in cancer metastasis patientspecimens. RNA was extracted from prostate cancer metastasis to lymphnode obtained from two different patients, as well as from normalbladder (NB), normal kidney (NK), normal lung (NL), normal breast (NBr),normal ovary (NO), and normal pancreas (NPa). Northern blots with 10 μgof total RNA/lane were probed with 205P1B5 sequence. Size standards inkilobases (kb) are indicated on the side. The results show expression of205P1B5 in both cancer metastasis samples but not in the normal tissuestested.

Example 5 Production of Recombinant 205P1B5 in Prokaryotic Systems

To express recombinant 205P1B5 in prokaryotic cells, the full or partiallength 205P1B5 cDNA sequences can be cloned into any one of a variety ofexpression vectors known in the art. One or more of the followingregions of 205P1B5 are expressed in these constructs, amino acids 1 to529; or any 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,.19, 20, 21, 22,23, 24,25, 26, 27, 28, 29, 30 or more contiguous amino acids from205P1B5, variants, or analogs thereof.

A. In Vitro Transcription and Translation Constructs:

pCRII:

To generate 205P1B5 sense and anti-sense RNA probes for RNA in situinvestigations, pCRII constructs (Invitrogen, Carlsbad Calif.) aregenerated encoding either all or fragments of the 205P1B5 cDNA. ThepCRII vector has Sp6 and T7 promoters flanking the insert to drive thetranscription of 205P1B5 RNA for use as probes in RNA in situhybridization experiments. These probes are used to analyze the cell andtissue expression of 205P1B5 at the RNA level. Transcribed 205P1B5 RNArepresenting the cDNA amino acid coding region of the 205P1B5 gene isused in in vitro translation systems such as the TnT™ CoupledReticulolysate System (Promega, Corp., Madison, Wis.) to synthesize205P1B5 protein.

B. Bacterial Constructs:

pGEX Constructs:

To generate recombinant 205P1B5 proteins in bacteria that are fused tothe Glutathione S-transferase (GST) protein, all or parts of the 205P1B5cDNA protein coding sequence are fused to the GST gene by cloning intopGEX-6P-1 or any other GST-fusion vector of the pGEX family (AmershamPharmacia Biotech, Piscataway, N.J.). These constructs allow controlledexpression of recombinant 205P1B5 protein sequences with GST fused atthe amino-terminus and a six histidine epitope (6× His) at thecarboxyl-terminus. The GST and 6× His tags permit purification of therecombinant fusion protein from induced bacteria with the appropriateaffinity matrix and allow recognition of the fusion protein withanti-GST and anti-His antibodies. The 6× His tag is generated by adding6 histidine codons to the cloning primer at the 3′ end, e.g., of theopen reading frame (ORF). A proteolytic cleavage site, such as thePreScission™ recognition site in pGEX-6P-1, may be employed such that itpermits cleavage of the GST tag from 205P1B5-related protein. Theampicillin resistance gene and pBR322 origin permits selection andmaintenance of the pGEX plasmids in E. coli.

pMAL Constructs:

To generate, in bacteria, recombinant 205P1B5 proteins that are fused tomaltose-binding protein (MBP), all or parts of the 205P?B5 cDNA proteincoding sequence are fused to the MBP gene by cloning into the pMAL-c2Xand pMAL-p2X vectors (New England Biolabs, Beverly, Mass.). Theseconstructs allow controlled expression of recombinant 205P1B5 proteinsequences with MBP fused at the amino-terminus and a 6× His epitope tagat the carboxyl-terminus. The MBP and 6× His tags permit purification ofthe recombinant protein from induced bacteria with the appropriateaffinity matrix and allow recognition of the fusion protein withanti-MBP and anti-His antibodies. The 6× His epitope tag is generated byadding 6 histidine codons to the 3′ cloning primer. A Factor Xarecognition site permits cleavage of the pMAL tag from 205P1B5. ThepMAL-c2X and pMAL-p2X vectors are optimized to express the recombinantprotein in the cytoplasm or periplasm respectively. Periplasm expressionenhances folding of proteins with disulfide bonds.

pET Constructs:

To express 205P1B5 in bacterial cells, all or parts of the 205P1B5 cDNAprotein coding sequence are cloned into the pET family of vectors(Novagen, Madison, Wis.). These vectors allow tightly controlledexpression of recombinant 205P1B5 protein in bacteria with and withoutfusion to proteins that enhance solubility, such as NusA and thioredoxin(Trx), and epitope tags, such as 6× His and S-Tag™ that aid purificationand detection of the recombinant protein. For example, constructs aremade utilizing pET NusA fusion system 43.1 such that regions of the205P1B5 protein are expressed as amino-terminal fusions to NusA.

C. Yeast Constructs:

pESC Constructs:

To express 205P1B5 in the yeast species Saccharomyces cerevisiae forgeneration of recombinant protein and functional studies, all or partsof the 205P1B5 cDNA protein coding sequence are cloned into the pESCfamily of vectors each of which contain 1 of 4 selectable markers, HIS3,TRP1, LEU2, and URA3 (Stratagene, La Jolla, Calif.). These vectors allowcontrolled expression from the same plasmid of up to 2 different genesor cloned sequences containing either Flag™ or Myc epitope tags in thesame yeast cell. This system is useful to confirm protein-proteininteractions of 205P1B5. In addition, expression in yeast yields similarpost-translational modifications, such as glycosylations andphosphorylations, that are found when expressed in eukaryotic cells.

pESP Constructs:

To express 205P1B5 in the yeast species Saccharomyces pombe, all orparts of the 205P1B5 cDNA protein coding sequence are cloned into thepESP family of vectors. These vectors allow controlled high level ofexpression of a 205P1B5 protein sequence that is fused at either theamino terminus or at the carboxyl terminus to GST which aidspurification of the recombinant protein. A Flag™ epitope tag allowsdetection of the recombinant protein with anti-Flag™ antibody.

Example 6 Production of Recombinant 205P1B5 in Eukaryotic Systems

A. Mammalian Constructs:

To express recombinant 205P1B5 in eukaryotic cells, the full or partiallength 205P1B5 cDNA sequences can be cloned into any one of a variety ofexpression vectors known in the art. One or more of the followingregions of 205P1B5 are expressed in these constructs, amino acids 1 to529; or any 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30 or more contiguous amino acids from205P1B5, variants, or analogs thereof.

The constructs can be transfected into any one of a wide variety ofmammalian cells such as 293T cells. Transfected 293T cell lysates can beprobed with the anti-205P1B5 polyclonal serum, described herein.

pcDNA4/HisMax Constructs:

To express 205P1B5 in mammalian cells, the 205P1B5 ORF, or portionsthereof, of 205P1B5 are cloned into pcDNA4/HisMax Version A (Invitrogen,Carlsbad, Calif.). Protein expression is driven from the cytomegalovirus(CMV) promoter and the SP16 translational enhancer. The recombinantprotein has Xpress™ and six histidine (6× His) epitopes fused to theamino-terminus. The pcDNA4/HisMax vector also contains the bovine growthhormone (BGH) polyadenylation signal and transcription terminationsequence to enhance mRNA stability along with the SV40 origin forepisomal replication and simple vector rescue in cell lines expressingthe large T antigen. The Zeocin resistance gene allows for selection ofmammalian cells expressing the protein and the ampicillin resistancegene and ColE1 origin permits selection and maintenance of the plasmidin E. coli.

pcDNA3.1/MycHis Constructs:

To express 205P1B5 in mammalian cells, the 205P1B5 ORF, or portionsthereof, of 205P1B5 with a consensus Kozak translation initiation siteare cloned into pcDNA3.1/MycHis Version A (Invitrogen, Carlsbad,Calif.). Protein expression is driven from the cytomegalovirus (CMV)promoter. The recombinant proteins have the myc epitope and 6× Hisepitope fused to the carboxyl-terminus. The pcDNA3.1/MycHis vector alsocontains the bovine growth hormone (BGH) polyadenylation signal andtranscription termination sequence to enhance mRNA stability, along withthe SV40 origin for episomal replication and simple vector rescue incell lines expressing the large T antigen. The Neomycin resistance genecan be used, as it allows for selection of mammalian cells expressingthe protein and the ampicillin resistance gene and ColE1 origin permitsselection and maintenance of the plasmid in E. coli.

pcDNA3.1/CT-GFP-TOPO Construct:

To express 205P1B5 in mammalian cells and to allow detection of therecombinant proteins using fluorescence, the 205P1B5 ORF, or portionsthereof, of 205P1B5 with a consensus Kozak translation initiation siteare cloned into pcDNA3.1/CT-GFP-TOPO (Invitrogen, Calif.). Proteinexpression is driven from the cytomegalovirus (CMV) promoter. Therecombinant proteins have the Green Fluorescent Protein (GFP) fused tothe carboxyl-terminus facilitating non-invasive, in vivo detection andcell biology studies. The pcDNA3.1CT-GFP-TOPO vector also contains thebovine growth hormone (BGH) polyadenylation signal and transcriptiontermination sequence to enhance mRNA stability along with the SV40origin for episomal replication and simple vector rescue in cell linesexpressing the large T antigen. The Neomycin resistance gene allows forselection of mammalian cells that express the protein, and theampicillin resistance gene and ColE1 origin permits selection andmaintenance of the plasmid in E. coli. Additional constructs with anamino-terminal GFP fusion are made in pcDNA3.1/NT-GFP-TOPO spanning theentire length of the 205P1B5 proteins.

PAPtap:

The 205P1B5 ORF, or portions thereof, of 205P1B5 are cloned intopAPtag-5 (GenHunter Corp. Nashville, Tenn.). This construct generates analkaline phosphatase fusion at the carboxyl-terminus of the 205P1B5proteins while fusing the IgGk signal sequence to the amino-terminus.Constructs are also generated in which alkaline phosphatase with anamino-terminal IgGk signal sequence is fused to the amino-terminus of205P1B5 proteins. The resulting recombinant 205P1B5 proteins areoptimized for secretion into the media of transfected mammalian cellsand can be used to identify proteins such as ligands or receptors thatinteract with the 205P1B5 proteins. Protein expression is driven fromthe CMV promoter and the recombinant proteins also contain myc and 6×His epitopes fused at the carboxyl-terminus that facilitates detectionand purification. The Zeocin resistance gene present in the vectorallows for selection of mammalian cells expressing the recombinantprotein and the ampicillin resistance gene permits selection of theplasmid in E. coli.

ptag5:

The 205P1B5 ORF, or portions thereof, of 205P1B5 are cloned into pTag-5.This vector is similar to pAPtag but without the alkaline phosphatasefusion. This construct generates 205P1B5 protein with an amino-terminalIgGk signal sequence and myc and 6× His epitope tags at thecarboxyl-terminus that facilitate detection and affinity purification.The resulting recombinant 205P1B5 protein is optimized for secretioninto the media of transfected mammalian cells, and is used as immunogenor ligand to identify proteins such as ligands or receptors thatinteract with the 205P1B5 proteins. Protein expression is driven fromthe CMV promoter. The Zeocin resistance gene present in the vectorallows for selection of mammalian cells expressing the protein, and theampicillin resistance gene permits selection of the plasmid in E. coli.

PsecFe:

The 205P1B5 ORF, or portions thereof, of 205P1B5 are also cloned intopsecFc. The psecFc vector was assembled by cloning the humanimmunoglobulin G1 (IgG) Fc (hinge, CH2, CH3 regions) into pSecTag2(Invitrogen, Calif.). This construct generates an IgG1 Fc fusion at thecarboxyl-terminus of the 205P1B5 proteins, while fusing the IgGK signalsequence to N-terminus. 205P1B5 fusions utilizing the murine IgG1 Fcregion are also used. The resulting recombinant 205P1B5 proteins areoptimized for secretion into the media of transfected mammalian cells,and can be used as immunogens or to identify proteins such as ligands orreceptors that interact with the 205P1B5 protein. Protein expression isdriven from the CMV promoter. The hygromycin resistance gene present inthe vector allows for selection of mammalian cells that express therecombinant protein, and the ampicillin resistance gene permitsselection of the plasmid in E. coli.

pSRα Constructs:

To generate mammalian cell lines that express 205P1B5 constitutively,205P1B5 ORF, or portions thereof, of 205P1B5 are cloned into pSRαconstructs. Amphotropic and ecotropic retroviruses are generated bytransfection of pSRα constructs into the 293T-10A1 packaging line orco-transfection of pSRα and a helper plasmid (containing deletedpackaging sequences) into the 293 cells, respectively. The retrovirus isused to infect a variety of mammalian cell lines, resulting in theintegration of the cloned gene, 205P1B5, into the host cell-lines.Protein expression is driven from a long terminal repeat (LTR). TheNeomycin resistance gene present in the vector allows for selection ofmammalian cells that express the protein, and the ampicillin resistancegene and ColE1 origin permit selection and maintenance of the plasmid inE. coli. The retroviral vectors can thereafter be used for infection andgeneration of various cell lines using, for example, PC3, NIH 3T3,TsuPr1, 293 or rat-1 cells.

Additional pSRα constructs are made that fuse an epitope tag such as theFLAG™ tag to the carboxyl-terminus of 205P1B5 sequences to allowdetection using anti-Flag antibodies. For example, the FLAG™ sequence 5′gat tac aag gat gac gac gat aag 3′ is added to cloning primer at the 3′end of the ORF. Additional pSRα constructs are made to produce bothamino-terminal and carboxyl-terminal GFP and myc/6× His fusion proteinsof the full-length 205P1B5 proteins.

Additional Viral Vectors:

Additional constructs are made for viral-mediated delivery andexpression of 205P1B5. High virus titer leading to high level expressionof 205P1B5 is achieved in viral delivery systems such as adenoviralvectors and herpes amplicon vectors. The 205P1B5 coding sequences orfragments thereof are amplified by PCR and subcloned into the AdEasyshuttle vector (Stratagene). Recombination and virus packaging areperformed according to the manufacturer's instructions to generateadenoviral vectors. Alternatively, 205P1B5 coding sequences or fragmentsthereof are cloned into the HSV-1 vector (Imgenex) to generate herpesviral vectors. The viral vectors are thereafter used for infection ofvarious cell lines such as PC3, NIH 3T3, 293 or rat-1 cells.

Regulated Expression Systems:

To control expression of 205P1B5 in mammalian cells, coding sequences of205P1B5, or portions thereof, are cloned into regulated mammalianexpression systems such as the T-Rex System (Invitrogen), the GeneSwitchSystem (Invitrogen) and the tightly-regulated Ecdysone System(Sratagene). These systems allow the study of the temporal andconcentration dependent effects of recombinant 205P1B5. These vectorsare thereafter used to control expression of 205P1B5 in various celllines such as PC3, NIH 3T3, 293 or rat-1 cells.

B. Baculovirus Expression Systems

To generate recombinant 205P1B5 proteins in a baculovirus expressionsystem, 205P1B5 ORF, or portions thereof, are cloned into thebaculovirus transfer vector pBlueBac 4.5 (Invitrogen), which provides aHis-tag at the N-terminus. Specifically, pBlueBac-205P1B5 isco-transfected with helper plasmid pBac-N-Blue (Invitrogen) into SF9(Spodoptera frugiperda) insect cells to generate recombinant baculovirus(see Invitrogen instruction manual for details). Baculovirus is thencollected from cell supernatant and purified by plaque assay.

Recombinant 205P1B5 protein is then generated by infection of HighFiveinsect cells (Invitrogen) with purified baculovirus. Recombinant 205P1B5protein can be detected using anti-205P1B5 or anti-His-tag antibody.205P1B5 protein can be purified and used in various cell-based assays oras immunogen to generate polyclonal and monoclonal antibodies specificfor 205P1B5.

Example 7 Antigenicity Profiles and Secondary Structure

FIG. 5, FIG. 6, FIG. 7, FIG. 8, and FIG. 9 depict graphically five aminoacid profiles of the 205P1B5 amino acid sequence, each assessmentavailable by accessing the ProtScale website (URL world wide web URLexpasy.ch/cgi-bin/protscale.pl) on the ExPasy molecular biology server.

These profiles: FIG. 5, Hydrophilicity, (Hopp T. P., Woods K. R., 1981.Proc. Natl. Acad. Sci. U.S.A. 78:3824-3828); FIG. 6, Hydropathicity,(Kyte J., Doolittle R. F., 1982. J. Mol. Biol. 157:105-132); FIG. 7,Percentage Accessible Residues (Janin J., 1979 Nature 277:491492); FIG.8, Average Flexibility, (Bhaskaran R., and Ponnuswamy P. K., 1988. Int.J. Pept. Protein Res. 32:242-255); FIG. 9, Beta-turn (Deleage, G., RouxB. 1987 Protein Engineering 1:289-294); and optionally others availablein the art, such as on the ProtScale website, were used to identifyantigenic regions of the 205P1B5 protein. Each of the above amino acidprofiles of 205P1B5 were generated using the following ProtScaleparameters for analysis: 1) A window size of 9; 2) 100% weight of thewindow edges compared to the window center; and, 3) amino acid profilevalues normalized to he between 0 and 1.

Hydrophilicity (FIG. 5), Hydropathicity (FIG. 6) and PercentageAccessible Residues (FIG. 7) profiles were used to determine stretchesof hydrophilic amino acids (i.e., values greater than 0.5 on theHydrophilicity and Percentage Accessible Residues profile, and valuesless than 0.5 on the Hydropathicity profile). Such regions are likely tobe exposed to the aqueous environment, be present on the surface of theprotein, and thus available for immune recognition, such as byantibodies.

Average Flexibility (FIG. 8) and Beta-turn (FIG. 9) profiles determinestretches of amino acids (i.e., values greater than 0.5 on the Beta-turnprofile and the Average Flexibility profile) that are not constrained insecondary structures such as beta sheets and alpha helices. Such regionsare also more likely to be exposed on the protein and thus accessible toimmune recognition, such as by antibodies.

Antigenic sequences of the 205P1B5 protein indicated, e.g., by theprofiles set forth in FIG. 5, FIG. 6, FIG. 7, FIG. 8, and/or FIG. 9 areused to prepare immunogens, either peptides or nucleic acids that encodethem, to generate therapeutic and diagnostic anti-205P1B5 antibodies.The immunogen can be any 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50 or more than 50contiguous amino acids, or the corresponding nucleic acids that encodethem from the 205P1B5 protein. In particular, peptide immunogens of theinvention can comprise, a peptide region of at least 5 amino acids ofFIG. 2 in any whole number increment up to 529 that includes an aminoacid position having a value greater than 0.5 in the Hydrophilicityprofile of FIG. 5; a peptide region of at least 5 amino acids of FIG. 2in any whole number increment up to 529 that includes an amino acidposition having a value less than 0.5 in the Hydropathicity profile ofFIG. 6; a peptide region of at least 5 amino acids of FIG. 2 in anywhole number increment up to 529 that includes an amino acid positionhaving a value greater than 0.5 in the Percent Accessible Residuesprofile of FIG. 7; a peptide region of at least 5 amino acids of FIG. 2in any whole number increment up to 529 that includes an amino acidposition having a value greater than 0.5 in the Average Flexibilityprofile on FIG. 8; and, a peptide region of at least 5 amino acids ofFIG. 2 in any whole number increment up to 529 that includes an aminoacid position having a value greater than 0.5 in the Beta-turn profileof FIG. 9. Peptide immunogens of the invention can also comprise nucleicacids that encode any of the forgoing.

All immunogens of the invention, peptide or nucleic acid, can beembodied in human unit dose form, or comprised by a composition thatincludes a pharmaceutical excipient compatible with human physiology.

The secondary structure of 205P1B5, namely the predicted presence andlocation of alpha helices, extended strands, and random coils, ispredicted from the primary amino acid sequence using theHNN-Hierarchical Neural Network method (Guermeur, 1997, world wide webURL /pbil.ibcp.fr/cgi-bin/npsa_automat.pl?page=npsa_nn.html), accessedfrom the ExPasy molecular biology server (world wide web URL:expasy.ch/tools/). The analysis indicates that 205P1B5 is composed41.40% alpha helix, 12.67% extended strand, and 45.94% random coil (FIG.14A).

Analysis for the potential presence of transmembrane domains in 205P1B5was carried out using a variety of transmembrane prediction algorithmsaccessed from the ExPasy molecular biology server (world wide web URLexpasy.ch/tools/). The programs predict the presence of 5 transmembranedomains in 205P1B5, consistent with the structure of a G-protein coupledreceptor. Shown graphically in FIG. 14 are the results of analysis usingthe TMpred (FIG. 14B) and TMHMM (FIG. 14C) prediction programs depictingthe location of the 5 transmembrane domains. The results of eachprogram, namely the amino acids encoding the transmembrane domains aresummarized in Table XXI.

Example 8 Generation of 205P1B5 Polyclonal Antibodies

Polyclonal antibodies can be raised in a mammal, for example, by one ormore injections of an immunizing agent and, if desired, an adjuvant.Typically, the immunizing agent and/or adjuvant will be injected in themammal by multiple subcutaneous or intraperitoneal injections. Inaddition to immunizing with the full length 205P1B5 protein, computeralgorithms are employed in design of immunogens that, based on aminoacid sequence analysis contain characteristics of being antigenic andavailable for recognition by the immune system of the immunized host(see the Example entitled “Antigenicity Profiles”). Such regions wouldbe predicted to be hydrophilic, flexible, in beta-turn conformations,and be exposed on the surface of the protein (see, e.g., FIG. 5, FIG. 6,FIG. 7, FIG. 8, or FIG. 9 for amino acid profiles that indicate suchregions of 205P1B5).

For example, 205P1B5 recombinant bacterial fusion proteins or peptidesencoding hydrophilic, flexible, beta-turn regions of the 205P1B5sequence, such as amino acids 23-63, are used as antigens to generatepolyclonal antibodies in New Zealand White rabbits. It is useful toconjugate the immunizing agent to a protein known to be immunogenic inthe mammal being immunized. Examples of such immunogenic proteinsinclude, but are not limited to, keyhole limpet hemocyanin (KLH), serumalbumin, bovine thyroglobulin, and soybean trypsin inhibitor. In oneembodiment, a peptide encoding amino acids 23-63 of 205P1B5 isconjugated to KLH and used to immunize the rabbit. Alternatively theimmunizing agent may include all or portions of the 205P1B5 protein,analogs or fusion proteins thereof. For example, the 205P1B5 amino acidsequence can be fused using recombinant DNA techniques to any one of avariety of fusion protein partners that are well known in the art, suchas glutathione-S-transferase (GST) and HIS tagged fusion proteins. Suchfusion proteins are purified from induced bacteria using the appropriateaffinity matrix.

In one embodiment, a GST-fusion protein encoding the predicted secondextracellular loop of 205P1B5 (amino acids 27-271) is produced andpurified and used as immunogen (see the section entitled “Production of205P1B5 in Prokaryotic Systems”). Other recombinant bacterial fusionproteins that may be employed include maltose binding protein, LacZ,thioredoxin, NusA, or an immunoglobulin constant region (see the sectionentitled “Production of 205P1B5 in Prokaryotic Systems” and CurrentProtocols In Molecular Biology, Volume 2, Unit 16, Frederick M. Ausubulet al. eds., 1995; Linsley, P. S., Brady, W., Urnes, M., Grosmaire, L.,Damle, N., and Ledbetter, L.(1991) J. Exp. Med. 174, 561-566).

In addition to bacterial derived fusion proteins, mammalian expressedprotein antigens are also used. These antigens are expressed frommammalian expression vectors such as the Tag5 and Fc-fusion vectors (seethe section entitled “Production of Recombinant 205P1B5 in EukaryoticSystems”), and retain post-translational modifications such asglycosylations found in native protein. In one embodiment, amino acids27-271 is cloned into the Tag5 mammalian secretion vector. Therecombinant protein is purified by metal chelate chromatography fromtissue culture supernatants of 293T cells stably expressing therecombinant vector. The purified Tag5 205P1B5 protein is then used asimmunogen.

During the immunization protocol, it is useful to mix or emulsify theantigen in adjuvants that enhance the immune response of the hostanimal. Examples of adjuvants include, but are not limited to, completeFreund's adjuvant (CFA) and MPL-TDM adjuvant (monophosphoryl Lipid A,synthetic trehalose dicorynomycolate).

In a typical protocol, rabbits are initially immunized subcutaneouslywith up to 200 μg, typically 100-200 μg, of fusion protein or peptideconjugated to KLH mixed in complete Freund's adjuvant (CFA). Rabbits arethen injected subcutaneously every two weeks with up to 200 μg,typically 100-200 μg, of the immunogen in incomplete Freund's adjuvant(IFA). Test bleeds are taken approximately 7-10 days following eachimmunization and used to monitor the titer of the antiserum by ELISA.

To test reactivity and specificity of immune serum, such as the rabbitserum derived from immunization with Tag5 205P1B5 encoding amino acids27-271, the full-length 205P1B5 cDNA is cloned into pCDNA 3.1 myc-hisexpression vector Invitrogen, see the Example entitled “Production ofRecombinant 205P1B5 in Eukaryotic Systems”). After transfection of theconstructs into 293T cells, cell lysates are probed with theanti-205P1B5 serum and with anti-His antibody (Santa CruzBiotechnologies, Santa Cruz, Calif.) to determine specific reactivity todenatured 205P1B5 protein using the Western blot technique.Immunoprecipitation and flow cytometric analyses of 293T and otherrecombinant 205P1B5-expressing cells determine recognition of nativeprotein by the antiserum. In addition, Western blot,immunoprecipitation, fluorescent microscopy, and flow cytometrictechniques using cells that endogenously express 205P1B5 are carried outto test specificity.

The anti-serum from the Tag5 205P1B5 immunized rabbit the serum isaffinity purified by passage over a column composed of the Tag5 antigencovalently coupled to Affigel matrix (BioRad, Hercules, Calif.). Theserum is then further purified by protein G affinity chromatography toisolate the IgG fraction. Serum from rabbits immunized with fusionproteins, such as GST and MBP fusion proteins, are purified by depletionof antibodies reactive to the fusion partner sequence by passage over anaffinity column containing the fusion partner either alone or in thecontext of an irrelevant fusion protein. Sera from other His-taggedantigens and peptide immunized rabbits as well as fusion partnerdepleted sera are affinity purified by passage over a column matrixcomposed of the original protein immunogen or free peptide.

Example 9 Generation of 205P1B5 Monoclonal Antibodies (mAbs)

In one embodiment, therapeutic mAbs to 205P1B5 comprise those that reactwith epitopes of the protein that would disrupt or modulate thebiological function of 205P1B5, for example those that would disrupt itsinteraction with ligands or proteins that mediate or are involved in itsbiological activity. Therapeutic mAbs also comprise those thatspecifically bind epitopes of 205P1B5 exposed on the cell surface andthus are useful in targeting mAb-toxin conjugates. Immunogens forgeneration of such mAbs include those designed to encode or contain theentire 205P1B5 protein or regions of the 205P1B5 protein predicted to beantigenic from computer analysis of the amino acid sequence (see, e.g.,FIG. 5, FIG. 6, FIG. 7, FIG. 8, or FIG. 9, and the Example entitled“Antigenicity Profiles”). Immunogens include peptides, recombinantbacterial proteins, and mammalian expressed Tag 5 proteins and human andmurine IgG FC fusion proteins. In addition, cells expressing high levelsof 205P1B5, such as 293T-205P1B5 cells, are used to immunize mice.

To generate mAbs to 205P1B5, mice are first immunized intraperitoneally(IP) with, typically, 10-50 μg of protein immunogen or10⁷205P1B5-expressing cells mixed in complete Freund's adjuvant. Miceare then subsequently immunized IP every 2-4 weeks with, typically,10-50 μg of protein immunogen or 10⁷ cells mixed in incomplete Freund'sadjuvant Alternatively, MPL-TDM adjuvant is used in immunizations. Inaddition to the above protein and cell-based immunization strategies, aDNA-based immunization protocol is employed in which a mammalianexpression vector encoding 205P1B5 sequence is used to immunize mice bydirect injection of the plasmid DNA. For example, the predicted secondextracellular loop of 205P1B5, amino acids 27-271, is cloned into theTag5 mammalian secretion vector and the recombinant vector is used asimmunogen. In another example, amino acids 23-63 (predicted to beantigenic from sequence analysis, see, e.g., FIG. 5, FIG. 6, FIG. 7,FIG. 8 or FIG. 9) is cloned into an Fc-fusion secretion vector in whichthe 205P1B5 sequence is fused at the amino-terminus to an IgK leadersequence and at the carboxyl-terminus to the coding sequence of thehuman IgG Fc region. This recombinant vector is then used as immunogen.The plasmid immunization protocols are used in combination with purifiedproteins expressed from the same vector and with cells expressing205P1B5.

During the immunization protocol, test bleeds are taken 7-10 daysfollowing an injection to monitor titer and specificity of the immuneresponse. Once appropriate reactivity and specificity is obtained asdetermined by ELISA, Western blotting, immunoprecipitation, fluorescencemicroscopy, and flow cytometric analyses, fusion and hybridomageneration is then carried out with established procedures well known inthe art (see, e.g., Harlow and Lane, 1988).

In one embodiment for generating 205P1B5 monoclonal antibodies, a Tag5205P1B5 antigen encoding amino acids 27-271 is expressed and purifiedfrom stably transfected 293T cells. Balb C mice are initially immunizedintraperitoneally with 25 μg of the Tag5 205P1B5 protein mixed incomplete Freund's adjuvant Mice are subsequently immunized every twoweeks with 25 μg of the antigen mixed in incomplete Freund's adjuvantfor a total of three immunizations. ELISA using the Tag5 antigendetermines the titer of serum from immunized mice. Reactivity andspecificity of serum to full length 205P1B5 protein is monitored byWestern blotting, immunoprecipitation and flow cytometry using 293Tcells transfected with an expression vector encoding the 205P1B5 cDNA(see e.g., the Example entitled “Production of Recombinant 205P1B5 inEukaryotic Systems”). Other recombinant 205P1B5-expressing cells orcells endogenously expressing 205P1B5 are also used. Mice showing thestrongest reactivity are rested and given a final injection of Tag5antigen in PBS and then sacrificed four days later. The spleens of thesacrificed mice are harvested and fused to SPO/2 myeloma cells usingstandard procedures (Harlow and Lane, 1988). Supernatants from HATselected growth wells are screened by ELISA, Western blot,immunoprecipitation, fluorescent microscopy, and flow cytometry toidentify 205P1B5 specific antibody-producing clones.

The binding affinity of a 205P1B5 monoclonal antibody is determinedusing standard technologies. Affinity measurements quantify the strengthof antibody to epitope binding and are used to help define which 205P1B5monoclonal antibodies preferred for diagnostic or therapeutic use, asappreciated by one of skill in the art. The BIAcore system (Uppsala,Sweden) is a preferred method for determining binding affinity. TheBIAcore system uses surface plasmon resonance (SPR, Welford K. 1991,Opt. Quant. Elect. 23:1; Morton and Myszka, 1998, Methods in Enzymology295: 268) to monitor biomolecular interactions in real time. BIAcoreanalysis conveniently generates association rate constants, dissociationrate constants, equilibrium dissociation constants, and affinityconstants.

Example 10 HLA Class I and Class II Binding Assays

HLA class I and class II binding assays using purified HLA molecules areperformed in accordance with disclosed protocols (e.g., PCT publicationsWO 94/20127 and WO 94/03205; Sidney et al., Current Protocols inImmunology 18.3.1 (1998); Sidney, et al., J. Immunol. 154:247 (1995);Sette, et al., Mol. Immunol. 31:813 (1994)). Briefly, purified MHCmolecules (5 to 500 nM) are incubated with various unlabeled peptideinhibitors and 1-10 nM ¹²⁵I-radiolabeled probe peptides as described.Following incubation, MHC-peptide complexes are separated from freepeptide by gel filtration and the fraction of peptide bound isdetermined. Typically, in preliminary experiments, each MHC preparationis titered in the presence of fixed amounts of radiolabeled peptides todetermine the concentration of HLA molecules necessary to bind 10-20% ofthe total radioactivity. All subsequent inhibition and direct bindingassays are performed using these HLA concentrations.

Since under these conditions [label]<[HLA] and IC₅₀≧[HLA], the measuredIC₅₀ values are reasonable approximations of the true K_(D) values.Peptide inhibitors are typically tested at concentrations ranging from120 μg/ml to 1.2 ng/ml, and are tested in two to four completelyindependent experiments. To allow comparison of the data obtained indifferent experiments, a relative binding figure is calculated for eachpeptide by dividing the IC₅₀ of a positive control for inhibition by theIC₅₀ for each tested peptide (typically unlabeled versions of theradiolabeled probe peptide). For database purposes, and inter-experimentcomparisons, relative binding values are compiled. These values cansubsequently be converted back into IC₅₀ nM values by dividing the IC₅₀nM of the positive controls for inhibition by the relative binding ofthe peptide of interest. This method of data compilation is accurate andconsistent for comparing peptides that have been tested on differentdays, or with different lots of purified MHC.

Binding assays as outlined above may be used to analyze HLA supermotifand/or HLA motif-bearing peptides.

Example 11 Identification of HLA Supermotif- and Motif-Bearing CTLCandidate Epitopes

HLA vaccine compositions of the invention can include multiple epitopes.The multiple epitopes can comprise multiple HLA supermotifs or motifs toachieve broad population coverage. This example illustrates theidentification and confirmation of supermotif- and motif-bearingepitopes for the inclusion in such a vaccine composition. Calculation ofpopulation coverage is performed using the strategy described below.

Computer Searches and Algorithms for Identification of Supermotif and/orMotif-Bearing Epitopes

The searches performed to identify the motif-bearing peptide sequencesin the Example entitled “Antigenicity Profiles” and Tables V-XVII employthe protein sequence data from the gene product of 205P1B5 set forth inFIGS. 2 and 3.

Computer searches for epitopes bearing HLA Class I or Class IIsupermotifs or motifs are performed as follows. All translated 205P1B5protein sequences are analyzed using a text string search softwareprogram to identify potential peptide sequences containing appropriateHLA binding motifs; such programs are readily produced in accordancewith information in the art in view of known motif/supermotifdisclosures. Furthermore, such calculations can be made mentally.

Identified A2-, A3-, and DR-supermotif sequences are scored usingpolynomial algorithms to predict their capacity to bind to specificHLA-Class I or Class II molecules. These polynomial algorithms accountfor the impact of different amino acids at different positions, and areessentially based on the premise that the overall affinity (or ΔG) ofpeptide-HLA molecule interactions can be approximated as a linearpolynomial function of the type:“ΔG”=a _(1i) ×a _(2i) ×a _(3i) , . . . ×a _(ni)where a_(ji) is a coefficient which represents the effect of thepresence of a given amino acid (f) at a given position (i) along thesequence of a peptide of n amino acids. The crucial assumption of thismethod is that the effects at each position are essentially independentof each other (i.e., independent binding of individual side-chains).When residue j occurs at position i in the peptide, it is assumed tocontribute a constant amount j_(i) to the free energy of binding of thepeptide irrespective of the sequence of the rest of the peptide.

The method of derivation of specific algorithm coefficients has beendescribed in Gulukota et al., J. Mol. Biol. 267:1258-126, 1997; (seealso Sidney et al., Human. Immunol. 45:79-93, 1996; and Southwood etal., J. Immunol. 160:3363-3373, 1998). Briefly, for all i positions,anchor and non-anchor alike, the geometric mean of the average relativebinding (ARB) of all peptides carrying j is calculated relative to theremainder of the group, and used as the estimate of j_(i). For Class IIpeptides, if multiple alignments are possible, only the highest scoringalignment is utilized, following an iterative procedure. To calculate analgorithm score of a given peptide in a test set, the ARB valuescorresponding to the sequence of the peptide are multiplied. If thisproduct exceeds a chosen threshold, the peptide is predicted to bind.Appropriate thresholds are chosen as a function of the degree ofstringency of prediction desired.

Selection of HLA-A2 Supertype Cross-Reactive Rentides

Complete protein sequences from 205P1B5 are scanned utilizing motifidentification software, to identify 8-, 9-, 10- and 11-mer sequencescontaining the HLA-A2-supermotif main anchor specificity. Typically,these sequences are then scored using the protocol described above andthe peptides corresponding to the positive-scoring sequences aresynthesized and tested for their capacity to bind purified HLA-A*0201molecules in vitro (HLA-A*0201 is considered a prototype A2 supertypemolecule).

These peptides are then tested for the capacity to bind to additionalA2-supertype molecules (A*0202, A*0203, A*0206, and A*6802). Peptidesthat bind to at least three of the five A2-supertype alleles tested aretypically deemed A2-supertype cross-reactive binders. Preferred peptidesbind at an affinity equal to or less than 500 nM to three or more HLA-A2supertype molecules.

Selection of HA-A3 Supermotif-Bearing Epitopes

The 205P1B5 protein sequence scanned above is also examined for thepresence of peptides with the HLA-A3-supermotif primary anchors.Peptides corresponding to the BLA A3 supermotif-bearing sequences arethen synthesized and tested for binding to HLA-A*0301 and HLA-A*1101molecules, the molecules encoded by the two most prevalent A3-supertypealleles. The peptides that bind at least one of the two alleles withbinding affinities of<500 nM, often<200 nM, are then tested for bindingcross-reactivity to the other common A3-supertype alleles (e.g., A*3101,A*3301, and A*6801) to identify those that can bind at least three ofthe five HLA-A3-supertype molecules tested.

Selection of HLA-B7 supermotif Bearing Epitopes

The 205P1B5 protein is also analyzed for the presence of 8-, 9-, 10-, or11-mer peptides with the HLA-B7-supermotif. Corresponding peptides aresynthesized and tested for binding to HLA-B*0702, the molecule encodedby the most common B7-supertype allele (i.e., the prototype B7 supertypeallele). Peptides binding B*0702 with IC5 of≦500 nM are identified usingstandard methods. These peptides are then tested for binding to othercommon B7-supertype molecules (e.g., B*3501, B*5 101, B*5301, andB*5401). Peptides capable of binding to three or more of the fiveB7-supertype alleles tested are thereby identified.

Selection of A1 and A24 Motif-Bearing Epitopes

To further increase population coverage, HLA-A1 and -A24 epitopes canalso be incorporated into vaccine compositions. An analysis of the205P1B5 protein can also be performed to identify HLA-A1- andA24-motif-containing sequences.

High affinity and/or cross-reactive binding epitopes that bear othermotif and/or supermotifs are identified using analogous methodology.

Example 12 Confirmation of Immunogenicity

Cross-reactive candidate CTL A2-supermotif-bearing peptides that areidentified as described herein are selected to confirm in vitroimmunogenicity. Confirmation is performed using the followingmethodology:

Target Cell Lines for Cellular Screening:

The 0.221A2.1 cell line, produced by transferring the HLA-A2.1 gene intothe HLA-A, -B, -C null mutant human B-lymphoblastoid cell line 721.221,is used as the peptide-loaded target to measure activity ofHLA-A2.1-restricted CTL. This cell line is grown in RPMI-1640 mediumsupplemented with antibiotics, sodium pyruvate, nonessential amino acidsand 10% (v/v) heat inactivated FCS. Cells that express an antigen ofinterest, or transfectants comprising the gene encoding the antigen ofinterest, can be used as target cells to confirm the ability ofpeptide-specific CTLs to recognize endogenous antigen.

Primary CTL Induction Cultures:

Generation of Dendritic Cells (DC):

PBMCs are thawed in RPMI with 30 μg/ml DNAse, washed twice andresuspended in complete medium (RPMI-1640 plus 5% AB human serum,nonessential amino acids, sodium pyruvate, L-glutamine andpenicillin/streptomycin). The monocytes are purified by plating 10×10⁶PBMC/well in a 6-well plate. After 2 hours at 37° C., the non-adherentcells are removed by gently shaking the plates and aspirating thesupernatants. The wells are washed a total of three times with 3 ml RPMIto remove most of the non-adherent and loosely adherent cells. Three mlof complete medium containing 50 ng/ml of GM-CSF and 1,000 U/ml of IL-4are then added to each well. TNFα is added to the DCs on day 6 at 75ng/ml and the cells are used for CTL induction cultures on day 7.

Induction of CTL with DC and Peptide:

CD8+ T-cells are isolated by positive selection with Dynalimmunomagnetic beads (Dynabeads® M450) and the detacha-bead® reagent.Typically about 200-250×10⁶ PBMC are processed to obtain 24×10⁶ CD8⁺T-cells (enough for a 48-well plate culture). Briefly, the PBMCs arethawed in RPMI with 30 μg/ml DNAse, washed once with PBS containing 1%human AB serum and resuspended in PBS/1% AB serum at a concentration of20×10⁶cells/ml. The magnetic beads are washed 3 times with PBS/AB serum,added to the cells (140 μl beads/20×10⁶ cells) and incubated for 1 hourat 4° C. with continuous mixing. The beads and cells are washed 4× withPBS/AB serum to remove the nonadherent cells and resuspended at 100×10⁶cells/ml (based on the original cell number) in PBS/AB serum containing100 μl/ml detacha-bead® reagent and 30 μg/ml DNAse. The mixture isincubated for 1 hour at room temperature with continuous mixing. Thebeads are washed again with PBS/AB/DNAse to collect the CD8+ T-cells.The DC are collected and centrifuged at 1300 rpm for 5-7 minutes, washedonce with PBS with 1% BSA, counted and pulsed with 40 μ/ml of peptide ata cell concentration of 1−2×10^(6/)ml in the presence of 3 μg/mlβ₂-microglobulin for 4 hours at 20° C. The DC are then irradiated (4,200rads), washed 1 time with medium and counted again.

Setting Up Induction Cultures:

0.25 ml cytokine-generated DC (at 1×10⁵ cells/ml) are co-cultured with0.25 ml of CD8+ T-cells (at 2×10⁶ cell/ml) in each well of a 48-wellplate in the presence of 10 ng/ml of IL7. Recombinant human IL-10 isadded the next day at a final concentration of 10 ng/ml and human IL-2is added 48 hours later at 10 IU/ml.

Restimulation of the Induction Cultures with Peptide-Pulsed AdherentCells:

Seven and fourteen days after the primary induction, the cells arerestimulated with peptide-pulsed adherent cells. The PBMCs are thawedand washed twice with RPMI and DNAse. The cells are resuspended at 5×10⁶cells/ml and irradiated at ˜4200 rads. The PBMCs are plated at 2×10⁶ in0.5 ml complete medium per well and incubated for 2 hours at 37° C. Theplates are washed twice with RPMI by tapping the plate gently to removethe nonadherent cells and the adherent cells pulsed with 10 μg/ml ofpeptide in the presence of 3 μg/ml β₂ microglobulin in 0.25 ml RPMI/5%AB per well for 2 hours at 37° C. Peptide solution from each well isaspirated and the wells are washed once with RPMI. Most of the media isaspirated from the induction cultures (CD8+ cells) and brought to 0.5 mlwith fresh media. The cells are then transferred to the wells containingthe peptide-pulsed adherent cells. Twenty four hours later recombinanthuman IL-10 is added at a final concentration of 10 ng/ml andrecombinant human IL2 is added the next day and again 2-3 days later at50 IU/ml (Tsai et al., Critical Reviews in Immunology 18(1-2):65-75,1998). Seven days later, the cultures are assayed for CTL activity in a⁵¹Cr release assay. In some experiments the cultures are assayed forpeptide-specific recognition in the in situ IFNγ ELISA at the time ofthe second restimulation followed by assay of endogenous recognition 7days later. After expansion, activity is measured in both assays for aside-by-side comparison.

Measurement of CTL Lytic Activity by ⁵¹Cr Release.

Seven days after the second restimulation, cytotoxicity is determined ina standard (5 hr) ⁵¹Cr release assay by assaying individual wells at asingle E:T. Peptide-pulsed targets are prepared by incubating the cellswith 10 μg/ml peptide overnight at 37° C.

Adherent target cells are removed from culture flasks with trypsin-EDTA.Target cells are labeled with 200 μCi of ⁵¹Cr sodium chromate (DupontWilmington, Del.) for 1 hour at 37° C. Labeled target cells aresuspended at 10⁶ per ml and diluted 1:10 with K562 cells at aconcentration of 3.3×10⁶/ml (an NK-sensitive erythroblastoma cell lineused to reduce non-specific lysis). Target cells (100 μl) and effectors(100 μl) are plated in 96 well round-bottomplates and incubated for 5hours at 37° C. At that time, 100 μl of supernatant are collected fromeach well and percent lysis is determined according to the formula:[(cpm of the test sample−cpm of the spontaneous ¹⁵Cr releasesample)/(cpm of the maximal ⁵¹Cr release sample−cpm of the spontaneous⁵¹Cr release sample)]×100.

Maximum and spontaneous release are determined by incubating the labeledtargets with 1% Triton X-100 and media alone, respectively. A positiveculture is defined as one in which the specific lysis(sample-background) is 10% or higher in the case of individual wells andis 15% or more at the two highest E:T ratios when expanded cultures areassayed.

In Situ Measurement of Human IFNγ Production as an Indicator ofPeptide-Specific and Endogenous Recognition

Immulon 2 plates are coated with mouse anti-human IFNγ monoclonalantibody (4 μg/ml 0.1M NaHCO₃, pH8.2) overnight at 4° C. The plates arewashed with Ca²⁺, Mg²⁺-free PBS/0.05% Tween 20 and blocked with PBS/100%FCS for two hours, after which the CTLs (100 μl/well) and targets (100μl/well) are added to each well, leaving empty wells for the standardsand blanks (which received media only). The target cells, eitherpeptide-pulsed or endogenous targets, are used at a concentration of1×10⁶ cells/ml. The plates are incubated for 48 hours at 37° C. with 5%CO₂.

Recombinant human IFN-gamma is added to the standard wells starting at400 pg or 1200 pg/100 microliter/well and the plate incubated for twohours at 37° C. The plates are washed and 100 μl of biotinylated mouseanti-human IFN-gamma monoclonal antibody (2 microgram/ml in PBS/3%FCS/0.05% Tween 20) are added and incubated for 2 hours at roomtemperature. After washing again, 100 microliter HRP-streptavidin(1:4000) are added and the plates incubated for one hour at roomtemperature. The plates are then washed 6× with wash buffer, 100microliter/well developing solution (TMB 1:1) are added, and the platesallowed to develop for 5-15 minutes. The reaction is stopped with 50microliter/well 1M H₃PO₄ and read at OD450. A culture is consideredpositive if it measured at least 50 pg of IFN-gamma/well abovebackground and is twice the background level of expression.

CTL Expansion.

Those cultures that demonstrate specific lytic activity againstpeptide-pulsed targets and/or tumor targets are expanded over a two weekperiod with anti-CD3. Briefly, 5×10⁴ CD8+ cells are added to a T75 flaskcontaining the following: 1×10⁶ irradiated (4,200 rad) PBMC (autologousor allogeneic) per ml, 2×10⁵ irradiated (8,000 rad) EBV-transformedcells per ml, and OKT3 (anti-CD3) at 30 ng per ml in RPMI-1640containing 10% (v/v) human AB serum, non-essential amino acids, sodiumpyruvate, 25 μM 2-mercaptoethanol; L-glutamine andpenicillin/streptomycin. Recombinant human IL2 is added 24 hours laterat a final concentration of 200 IU/ml and every three days thereafterwith fresh media at 50 IU/ml. The cells are split if the cellconcentration exceeds 1×10⁶/ml and the cultures are assayed between days13 and 15 at E:T ratios of 30, 10, 3 and 1:1 in the ⁵¹Cr release assayor at 1×10⁶/ml in the in situ IFNγ assay using the same targets asbefore the expansion.

Cultures are expanded in the absence of anti-CD3⁺ as follows. Thosecultures that demonstrate specific lytic activity against peptide andendogenous targets are selected and 5×10⁴ CD8⁺ cells are added to a T25flask containing the following: 1×10⁶ autologous PBMC per ml which havebeen peptide-pulsed with 10 μg/ml peptide for two hours at 37° C. andirradiated (4,200 rad); 2×10⁵ irradiated (8,000 rad) EBV-transformedcells per ml RPMI-1640 containing 10%(v/v) human AB serum, non-essentialAA, sodium pyruvate, 25 mM 2-ME, L-glutamine and gentamicin.

Immunogenicity of A2 Supermotif-Bearing Peptides

A2-supermotif cross-reactive binding peptides are tested in the cellularassay for the ability to induce peptide-specific CTL in normalindividuals. In this analysis, a peptide is typically considered to bean epitope if it induces peptide-specific CTLs in at least individuals,and preferably, also recognizes the endogenously expressed peptide.

Immunogenicity can also be confirmed using PBMCs isolated from patientsbearing a tumor that expresses 205P1B5. Briefly, PBMCs are isolated frompatients, re-stimulated with peptide-pulsed monocytes and assayed forthe ability to recognize peptide-pulsed target cells as well astransfected cells endogenously expressing the antigen.

Evaluation of A*03/A11 Immunogenicity

HLA-A3 supermotif-bearing cross-reactive binding peptides are alsoevaluated for immunogenicity using methodology analogous for that usedto evaluate the immunogenicity of the HLA-A2 supermotif peptides.

Evaluation of B7 Immunogenicity

Immunogenicity screening of the B7-supertype cross-reactive bindingpeptides identified as set forth herein are confirmed in a manneranalogous to the confirmation of A2-and A3-supermotif-bearing peptides.

Peptides bearing other supermotifs/motifs, e.g. HLA-A1, HLA-A24 etc. arealso confirmed using similar methodology

Example 13 Implementation of the Extended Supermotif to Improve theBinding Capacity of Native Epitopes by Creating Analogs

HLA motifs and supermotifs (comprising primary and/or secondaryresidues) are useful in the identification and preparation of highlycross-reactive native peptides, as demonstrated herein. Moreover, thedefinition of HLA motifs and supermotifs also allows one to engineerhighly cross-reactive epitopes by identifying residues within a nativepeptide sequence which can be analoged to confer upon the peptidecertain characteristics, e.g. greater cross-reactivity within the groupof HLA molecules that comprise a supertype, and/or greater bindingaffinity for some or all of those HLA molecules. Examples of analogingpeptides to exhibit modulated binding affinity are set forth in thisexample.

Analoging at Primary Anchor Residues

Peptide engineering strategies are implemented to further increase thecross-reactivity of the epitopes. For example, the main anchors ofA2-supermotif-bearing peptides are altered, for example, to introduce apreferred L, I, V, or M at position 2, and I or V at the C-terminus.

To analyze the cross-reactivity of the analog peptides, each engineeredanalog is initially tested for binding to the prototype A2 supertypeallele A*0201, then, if A*0201 binding capacity is maintained, forA2-supertype cross-reactivity.

Alternatively, a peptide is confirmed as binding one or all supertypemembers and then analogued to modulate binding affinity to any one (ormore) of the supertype members to add population coverage.

The selection of analogs for immunogenicity in a cellular screeninganalysis is typically further restricted by the capacity of the parentwild type (WT) peptide to bind at least weakly, i.e., bind at an IC₅₀ of5000 nM or less, to three of more A2 supertype alleles. The rationalefor this requirement is that the WT peptides must be presentendogenously in sufficient quantity to be biologically relevant Analogedpeptides have been shown to have increased immunogenicity andcross-reactivity by T cells specific for the parent epitope (see, e.g.,Parkhurst et al., J. Immunol. 157:2539, 1996; and Pogue et al., Proc.Natl. Acad. Sci. USA 92:8166, 1995).

In the cellular screening of these peptide analogs, it is important toconfirm that analog-specific CTLs are also able to recognize thewild-type peptide and, when possible, target cells that endogenouslyexpress the epitope.

Analoging of HLA-A3 and B7-Supermotif-Bearing Peptides

Analogs of HLA-A3 supermotif-bearing epitopes are generated usingstrategies similar to those employed in analoging HLA-A2supermotif-bearing peptides. For example, peptides binding to 3/5 of theA3-supertype molecules are engineered at primary anchor residues topossess a preferred residue (V, S, M, or A) at position 2.

The analog peptides are then tested for the ability to bind A*03 andA*11 (prototype A3 supertype alleles). Those peptides thatdemonstrate≦500 nM binding capacity are then confirmed as havingA3-supertype cross-reactivity.

Similarly to the A2- and A3-motif bearing peptides, peptides binding 3or more B7-supertype alleles can be improved, where possible, to achieveincreased cross-reactive binding or greater binding affinity or bindinghalf life. B7 supermotif-bearing peptides are, for example, engineeredto possess a preferred residue (V, I, L, or F) at the C-terminal primaryanchor position, as demonstrated by Sidney et al. (J. Immunol.157:3480-3490, 1996).

Analoging at primary anchor residues of other motif and/orsupermotif-bearing epitopes is performed in a like manner.

The analog peptides are then be confirmed for immunogenicity, typicallyin a cellular screening assay. Again, it is generally important todemonstrate that analog-specific CTLs are also able to recognize thewild-type peptide and, when possible, targets that endogenously expressthe epitope.

Analoging at Secondary Anchor Residues

Moreover, HLA supermotifs are of value in engineering highlycross-reactive peptides and/or peptides that bind HLA molecules withincreased affinity by identifying particular residues at secondaryanchor positions that are associated with such properties. For example,the binding capacity of a B7 supermotif-bearing peptide with an Fresidue at position 1 is analyzed. The peptide is then analoged to, forexample, substitute L for F at position 1. The analoged peptide isevaluated for increased binding affinity, binding half life and/orincreased cross-reactivity. Such a procedure identifies analogedpeptides with enhanced properties.

Engineered analogs with sufficiently improved binding capacity orcross-reactivity can also be tested for immunogenicity inHLA-B7-transgenic mice, following for example, IFA immunization orlipopeptide immunization. Analogued peptides are additionally tested forthe ability to stimulate a recall response using PBMC from patients with205P1B5-expressing tumors.

Other Analoging, Strategies

Another form of peptide analoging, unrelated to anchor positions,involves the substitution of a cysteine with α-amino butyric acid. Dueto its chemical nature, cysteine has the propensity to form disulfidebridges and sufficiently alter the peptide structurally so as to reducebinding capacity. Substitution of α-amino butyric acid for cysteine notonly alleviates this problem, but has been shown to improve binding andcrossbinding capabilities in some instances (see, e.g., the review bySette et al., In: Persistent Viral Infections, Eds. R Ahmed and I. Chen,John Wiley & Sons, England, 1999).

Thus, by the use of single amino acid substitutions, the bindingproperties and/or cross-reactivity of peptide ligands for HLA supertypemolecules can be modulated.

Example 14 Identification and Confirmation of 205P1B5-Derived Sequenceswith HLA-DR Binding Motifs

Peptide epitopes bearing an HLA class II supermotif or motif areidentified and confirmed as outlined below using methodology similar tothat described for HLA Class I peptides.

Selection of HLA-DR-Supermotif-Bearing Epitopes.

To identify 205P1B5-derived, HLA class II HTL epitopes, the 205P1B5antigen is analyzed for the presence of sequences bearing anHLA-DR-motif or supermotif. Specifically, 15-mer sequences are selectedcomprising a DR-supermotif, comprising a 9-mer core, and three-residueN- and C-terminal flanking regions (15 amino acids total).

Protocols for predicting peptide binding to DR molecules have beendeveloped (Southwood et al., J. Immunol. 160:3363-3373, 1998). Theseprotocols, specific for individual DR molecules, allow the scoring, andranking, of 9-mer core regions. Each protocol not only scores peptidesequences for the presence of DR-supermotif primary anchors (i.e., atposition 1 and position 6) within a 9-mer core, but additionallyevaluates sequences for the presence of secondary anchors. Usingallele-specific selection tables (see, e.g., Southwood et al., ibid.),it has been found that these protocols efficiently select peptidesequences with a high probability of binding a particular DR molecule.Additionally, it has been found that performing these protocols intandem, specifically those for DR1, DR4w4, and DR7, can efficientlyselect DR cross-reactive peptides.

The 205P1B5-derived peptides identified above are tested for theirbinding capacity for various common HLA-DR molecules. All peptides areinitially tested for binding to the DR molecules in the primary panel:DR1, DR4w4, and DR7. Peptides binding at least two of these three DRmolecules are then tested for binding to DR2w2 β1, DR2w2 β2, DR6w19, andDR9 molecules in secondary assays. Finally, peptides binding at leasttwo of the four secondary panel DR molecules, and thus cumulatively atleast four of seven different DR molecules, are screened for binding toDR4w15, DR5w11, and DR8w2 molecules in tertiary assays. Peptides bindingat least seven of the ten DR molecules comprising the primary,secondary, and tertiary screening assays are considered cross-reactiveDR binders. 205P1B5-derived peptides found to bind common HLA-DR allelesare of particular interest.

Selection of DR3 Motif Peptides

Because HLA-DR3 is an allele that is prevalent in Caucasian, Black, andHispanic populations, DR3 binding capacity is a relevant criterion inthe selection of HTL epitopes. Thus, peptides shown to be candidates mayalso be assayed for their DR3 binding capacity. However, in view of thebinding specificity of the DR3 motif, peptides binding only to DR3 canalso be considered as candidates for inclusion in a vaccine formulation.

To efficiently identify peptides that bind DR3, target 205P1B5 antigensare analyzed for sequences carrying one of the two DR3-specific bindingmotifs reported by Geluk et al. (J. Immunol. 152:5742-5748, 1994). Thecorresponding peptides are then synthesized and confirmed as having theability to bind DR3 with an affinity of 1 μM or better, i.e., less than1 μM. Peptides are found that meet this binding criterion and qualify asHLA class II high affinity binders.

DR3 binding epitopes identified in this manner are included in vaccinecompositions with DR supermotif-bearing peptide epitopes.

Similarly to the case of HLA class I motif-bearing peptides, the classII motif-bearing peptides are analoged to improve affinity orcross-reactivity. For example, aspartic acid at position 4 of the 9-mercore sequence is an optimal residue for DR3 binding, and substitutionfor that residue often improves DR 3 binding.

Example 15 Immunogenicity of 20SP1B5 Derived HTL Epitopes

This example determines immunogenic DR supermotif- and DR3 motif-bearingepitopes among those identified using the methodology set forth herein.

Immunogenicity of HTL epitopes are confirmed in a manner analogous tothe determination of immunogenicity of CTL epitopes, by assessing theability to stimulate HTL responses and/or by using appropriatetransgenic mouse models. Immunogenicity is determined by screening for:1.) in vitro primary induction using normal PBMC or 2.) recall responsesfrom patients who have 205P1B5-expressing tumors.

Example 16 Calculation of Phenotypic Frequencies of HLA-Supertypes inVarious Ethnic Backgrounds to Determine Breadth of Population Coverage

This example illustrates the assessment of the breadth of populationcoverage of a vaccine composition comprised of multiple epitopescomprising multiple supermotifs and/or motifs.

In order to analyze population coverage, gene frequencies of HLA allelesare determined. Gene frequencies for each HLA allele are calculated fromantigen or allele frequencies utilizing the binomial distributionformulae gf=1−(SQRT(1−af)) (see, e.g., Sidney et al. Human Immunol.45:79-93, 1996). To obtain overall phenotypic frequencies, cumulativegene frequencies are calculated, and the cumulative antigen frequenciesderived by the use of the inverse formula [af=1−(1−Cgf)²].

Where frequency data is not available at the level of DNA typing,correspondence to the serologically defined antigen frequencies isassumed. To obtain total potential supertype population coverage nolinkage disequilibrium is assumed, and only alleles confirmed to belongto each of the supertypes are included (minimal estimates). Estimates oftotal potential coverage achieved by inter-loci combinations are made byadding to the A coverage the proportion of the non-A covered populationthat could be expected to be covered by the B alleles considered (e.g.,total=A+B*(1−A)). Confirmed members of the A3-like supertype are A3,A11, A31, A*3301, and A*6801. Although the A3-like supertype may alsoinclude A34, A66, and A*7401, these alleles were not included in overallfrequency calculations. Likewise, confirmed members of the A2-likesupertype family are A*0201, A*0202, A*0203, A*0204, A*0205, A*0206,A*0207, A*6802, and A*6901. Finally, the B7-like supertype-confirmedalleles are: B7, B*3501-03, B51, B*5301, B*5401, B*5501-2, B*5601,B*6701, and B*7801 (potentially also B*1401, B*3504-06, B*4201, andB*5602).

Population coverage achieved by combining the A2-, A3- and B7-supertypesis approximately 86% in five major ethnic groups. Coverage may beextended by including peptides bearing the A1 and A24 motifs. Onaverage, A1 is present in 12% and A24 in 29% of the population acrossfive different major ethnic groups (Caucasian, North American Black,Chinese, Japanese, and Hispanic). Together, these alleles arerepresented with an average frequency of 39% in these same ethnicpopulations. The total coverage across the major ethnicities when A1 andA24 are combined with the coverage of the A2-, A3- and B7-supertypealleles is>95%. An analogous approach can be used to estimate populationcoverage achieved with combinations of class II motif-bearing epitopes.

Immunogenicity studies in humans (e.g., Bertoni et al., J. Clin. Invest.100:503, 1997; Doolan et al., Immunity 7:97, 1997; and Threlkeld et al.,J. Immunol. 159:1648, 1997) have shown that highly cross-reactivebinding peptides are almost always recognized as epitopes. The use ofhighly cross-reactive binding peptides is an important selectioncriterion in identifying candidate epitopes for inclusion in a vaccinethat is immunogenic in a diverse population.

With a sufficient number of epitopes (as disclosed herein and from theart), an average population coverage is predicted to be greater than 95%in each of five major ethnic populations. The game theory Monte Carlosimulation analysis, which is known in the art (see e.g., Osborne, M. J.and Rubinstein, A. “A course in game theory” MIT Press, 1994), can beused to estimate what percentage of the individuals in a populationcomprised of the Caucasian, North American Black, Japanese, Chinese, andHispanic ethnic groups would recognize the vaccine epitopes describedherein. A preferred percentage is 90%. A more preferred percentage is95%.

Example 17 CTL Recognition of Endogenously Processed Antigens AfterPriming

This example confirms that CTL induced by native or analoged peptideepitopes identified and selected as described herein recognizeendogenously synthesized, i.e., native antigens.

Effector cells isolated from transgenic mice that are immunized withpeptide epitopes, for example HLA-A2 supermotif-bearing epitopes, arere-stimulated in vitro using peptide-coated stimulator cells. Six dayslater, effector cells are assayed for cytotoxicity and the cell linesthat contain peptide-specific cytotoxic activity are furtherre-stimulated. An additional six days later, these cell lines are testedfor cytotoxic activity on ⁵¹Cr labeled Jurkat-A2.1/K^(b) target cells inthe absence or presence of peptide, and also tested on ⁵¹Cr labeledtarget cells bearing the endogenously synthesized antigen, i.e. cellsthat are stably transfected with 205P1B5 expression vectors.

The results demonstrate that CTL lines obtained from animals primed withpeptide epitope recognize endogenously synthesized 205P1B5 antigen. Thechoice of transgenic mouse model to be used for such an analysis dependsupon the epitope(s) that are being evaluated. In addition toHLA-A*0201/K^(b) transgenic mice, several other transgenic mouse modelsincluding mice with human A11, which may also be used to evaluate A3epitopes, and B7 alleles have been characterized and others (e.g.transgenic mice for HLA-A1 and A24) are being developed. HLA-DR1 andHLA-DR3 mouse models have also been developed, which may be used toevaluate HTL epitopes.

Example 18 Activity of CTL-HTL Conjugated Epitopes in Transgenic Mice

This example illustrates the induction of CTLs and HTLs in transgenicmice, by use of a 205P1B5-derived CTL and HTL peptide vaccinecompositions. The vaccine composition used herein comprise peptides tobe administered to a patient with a 205P1B5-expressing tumor. Thepeptide composition can comprise multiple CTL and/or HTL epitopes. Theepitopes are identified using methodology as described herein. Thisexample also illustrates that enhanced immunogenicity can be achieved byinclusion of one or more HTL epitopes in a CTL vaccine composition; sucha peptide composition can comprise an HTL epitope conjugated to a CTLepitope. The CTL epitope can be one that binds to multiple HLA familymembers at an affinity of 500 nM or less, or analogs of that epitope.The peptides may be lipidated, if desired.

Immunization procedures:

Immunization of transgenic mice is performed as described (Alexander etal., J. Immunol. 159:4753-4761, 1997). For example, A2/K^(b) mice, whichare transgenic for the human HLA A2.1 allele and are used to confirm theimmunogenicity of HLA-A*0201 motif- or HLA-A2 supermotif-bearingepitopes, and are primed subcutaneously (base of the tail) with a 0.1 mlof peptide in Incomplete Freund's Adjuvant, or if the peptidecomposition is a lipidated CTL/HTL conjugate, in DMSO/saline, or if thepeptide composition is a polypeptide, in PBS or Incomplete Freund'sAdjuvant Seven days after priming, splenocytes obtained from theseanimals are restimulated with syngenic irradiated LPS-activatedlymphoblasts coated with peptide.

Cell Lines:

Target cells for peptide-specific cytotoxicity assays are Jurkat cellstransfected with the HLA-A2.1/K^(b) chimeric gene (e.g., Vitiello etal., J. Exp. Med 173:1007, 1991)

In Vitro CTL Activation:

One week after priming, spleen cells (30×10⁶ cells/flask) areco-cultured at 37° C. with syngeneic, irradiated (3000 rads), peptidecoated lymphoblasts (10×10⁶ cells/flask) in 10 ml of culture medium/T25flask. After six days, effector cells are harvested and assayed forcytotoxic activity.

Assay for Cytotoxic Activity:

Target cells (1.0 to 1.5×10⁶) are incubated at 37° C. in the presence of200 μl of ⁵¹Cr. After 60 minutes, cells are washed three times andresuspended in R10 medium. Peptide is added where required at aconcentration of 1 μg/ml. For the assay, 10⁴ ⁵¹Cr-labeled target cellsare added to different concentrations of effector cells (final volume of200 μL) in U-bottom 96-well plates. After a six hour incubation periodat 37° C., a 0.1 ml aliquot of supernatant is removed from each well andradioactivity is determined in a Micromedic automatic gamma counter. Thepercent specific lysis is determined by the formula: percent specificrelease=100×(experimental release−spontaneous release)/(maximumrelease−spontaneous release). To facilitate comparison between separateCTL assays run under the same conditions, % ⁵¹Cr release data isexpressed as lytic units/10⁶ cells. One lytic unit is arbitrarilydefined as the number of effector cells required to achieve 30% lysis of10,000 target cells in a six hour ⁵¹Cr release assay. To obtain specificlytic units/10⁶, the lytic units/10⁶ obtained in the absence of peptideis subtracted from the lytic units/10⁶ obtained in the presence ofpeptide. For example, if 30% ⁵¹Cr release is obtained at the effector(E): target (7) ratio of 50:1 (i.e., 5×10⁵ effector cells for 10,000targets) in the absence of peptide and 5:1 (i.e., 5×10⁴ effector cellsfor 10,000 targets) in the presence of peptide, the specific lytic unitswould be: [(1/50,000)−(1/500,000)]×10⁶=18 LU.

The results are analyzed to assess the magnitude of the CTL responses ofanimals injected with the immunogenic CTL/HTL conjugate vaccinepreparation and are compared to the magnitude of the CTL responseachieved using, for example, CTL epitopes as outlined above in theExample entitled “Confirmation of Immunogenicity”. Analyses similar tothis may be performed to confirm the immunogenicity of peptideconjugates containing multiple CTL epitopes and/or multiple HTLepitopes. In accordance with these procedures, it is found that a CTLresponse is induced, and concomitantly that an HTL response is inducedupon administration of such compositions.

Example 19 Selection of CTL and HTL Epitopes for Inclusion in an205P1B5-Specific Vaccine

This example illustrates a procedure for selecting peptide epitopes forvaccine compositions of the invention. The peptides in the compositioncan be in the form of a nucleic acid sequence, either single or one ormore sequences (i.e., minigene) that encodes peptide(s), or can besingle and/or polyepitopic peptides.

The following principles are utilized when selecting a plurality ofepitopes for inclusion in a vaccine composition. Each of the followingprinciples is balanced in order to make the selection.

Epitopes are selected which, upon administration, mimic immune responsesthat are correlated with 205P1B5 clearance. The number of epitopes useddepends on observations of patients who spontaneously clear 205P1B5. Forexample, if it has been observed that patients who spontaneously clear205P1B5 generate an immune response to at least three (3) from 205P1B5antigen, then three or four (3-4) epitopes should be included for HLAclass I. A similar rationale is used to determine HLA class II epitopes.

Epitopes are often selected that have a binding affinity of an IC₅₀ of500 nM or less for an HLA class I molecule, or for class II, an IC₅₀ of1000 nM or less; or HLA Class I peptides with high binding scores fromthe BIMAS web site, at URL bimas.dcrt.nih.gov/.

In order to achieve broad coverage of the vaccine through out a diversepopulation, sufficient supermotif bearing peptides, or a sufficientarray of allele-specific motif bearing peptides, are selected to givebroad population coverage. In one embodiment, epitopes are selected toprovide at least 80% population coverage. A Monte Carlo analysis, astatistical evaluation known in the art can be employed to assessbreadth, or redundancy, of population coverage.

When creating polyepitopic compositions, or a minigene that encodessame, it is typically desirable to generate the smallest peptidepossible that encompasses the epitopes of interest. The principlesemployed are similar, if not the same, as those employed when selectinga peptide comprising nested epitopes. For example, a protein sequencefor the vaccine composition is selected because it has maximal number ofepitopes contained within the sequence, i.e., it has a highconcentration of epitopes. Epitopes maybe nested or overlapping (i.e.,frame shifted relative to one another). For example, with overlappingepitopes, two 9-mer epitopes and one 10-mer epitope can be present in a10 amino acid peptide. Each epitope can be exposed and bound by an HLAmolecule upon administration of such a peptide. A multi-epitopic,peptide can be generated synthetically, recombinantly, or via cleavagefrom the native source. Alternatively, an analog can be made of thisnative sequence, whereby one or more of the epitopes comprisesubstitutions that alter the cross-reactivity and/or binding affinityproperties of the polyepitopic peptide. Such a vaccine composition isadministered for therapeutic or prophylactic purposes. This embodimentprovides for the possibility that an as yet undiscovered aspect ofimmune system processing will apply to the native nested sequence andthereby facilitate the production of therapeutic or prophylactic immuneresponse-inducing vaccine compositions. Additionally such an embodimentprovides for the possibility of motif-bearing epitopes for an HLA makeupthat is presently unknown. Furthermore, this embodiment (absent thecreating of any analogs) directs the immune response to multiple peptidesequences that are actually present in 205P1B5, thus avoiding the needto evaluate any junctional epitopes. Lastly, the embodiment provides aneconomy of scale when producing nucleic acid vaccine compositions.Related to this embodiment, computer programs can be derived inaccordance with principles in the art, which identify in a targetsequence, the greatest number of epitopes per sequence length

A vaccine composition comprised of selected peptides, when administered,is safe, efficacious, and elicits an immune response similar inmagnitude to an immune response that controls or clears cells that bearor overexpress 205P1B5.

Example 20 Construction of “Minigene” Multi-Epitope DNA Plasmids

This example discusses the construction of a minigene expressionplasmid. Minigene plasmids may, of course, contain variousconfigurations of B cell CTL and/or HTL epitopes or epitope analogs asdescribed herein.

A minigene expression plasmid typically includes multiple CTL and HTLpeptide epitopes. In the present example, HLA-A2, -A3,-B7supermotif-bearing peptide epitopes and HLA-A1 and -A24 motif-bearingpeptide epitopes are used in conjunction with DR supermotif-bearingepitopes and/or DR3 epitopes. HLA class I supermotif or motif-bearingpeptide epitopes derived 205P1B5, are selected such that multiplesupermotifs/motifs are represented to ensure broad population coverage.Similarly, HLA class II epitopes are selected from 205P1B5 to providebroad population coverage, i.e. both HLA DR-1-4-7 supermotif-bearingepitopes and HLA DR-3 motif-bearing epitopes are selected for inclusionin the minigene construct. The selected CTL and HTL epitopes are thenincorporated into a minigene for expression in an expression vector.

Such a construct may additionally include sequences that direct the HTLepitopes to the endoplasmic reticulum. For example, the Ii protein maybe fused to one or more HTL epitopes as described in the art, whereinthe CLIP sequence of the Ii protein is removed and replaced with an HLAclass II epitope sequence so that HLA class II epitope is directed tothe endoplasmic reticulum, where the epitope binds to an HLA class Hmolecules.

This example illustrates the methods to be used for construction of aminigene-bearing expression plasmid. Other expression vectors that maybe used for minigene compositions are available and known to those ofskill in the art

The minigene DNA plasmid of this example contains a consensus Kozaksequence and a consensus murine kappa Ig-light chain signal sequencefollowed by CTL and/or HTL epitopes selected in accordance withprinciples disclosed herein. The sequence encodes an open reading framefused to the Myc and His antibody epitope tag coded for by the pcDNA 3.1Myc-His vector.

Overlapping oligonucleotides that can, for example, average about 70nucleotides in length with 15 nucleotide overlaps, are synthesized andHPLC-purified, The oligonucleotides encode the selected peptide epitopesas well as appropriate linker nucleotides, Kozak sequence, and signalsequence. The final multiepitope minigene is assembled by extending theoverlapping oligonucleotides in three sets of reactions using PCR APerkin/Elmer 9600 PCR machine is used and a total of 30 cycles areperformed using the following conditions: 95° C. for 15 sec, annealingtemperature (5° below the lowest calculated Tm of each primer pair) for30 sec, and 72° C. for 1 min.

For example, a minigene is prepared as follows. For a first PCRreaction, 5 μg of each of two oligonucleotides are annealed andextended: In an example using eight oligonucleotides, i.e., four pairsof primers, oligonucleotides 1+2, 3+4, 5+6, and 7+8 are combined in 100μl reactions containing Pfu polymerase buffer (1×=10 mM KCL, 10 mM(NH4)₂SO₄, 20 mM Tris-chloride, pH 8.75, 2 mM MgSO₄, 0.1% Triton X-100,100 μg/ml BSA), 0.25 mM each dNTP, and 2.5 U of Pfu polymerase. Thefull-length dimer products are gel-purified, and two reactionscontaining the product of 1+2 and 3+4, and the product of 5+6 and 7+8are mixed, annealed, and extended for 10 cycles. Half of the tworeactions are then mixed, and 5 cycles of annealing and extensioncarried out before flanking primers are added to amplify the full lengthproduct. The fill-length product is gel-purified and cloned intopCR-blunt (Invitrogen) and individual clones are screened by sequencing.

Example 21 The Plasmid Construct and the Degree to which it InducesImmunogenicity

The degree to which a plasmid construct, for example a plasmidconstructed in accordance with the previous Example, is able to induceimmunogenicity is confirmed in vitro by determining epitope presentationby APC following transduction or transfection of the APC with anepitope-expressing nucleic acid construct Such a study determines“antigenicity” and allows the use of human APC. The assay determines theability of the epitope to be presented by the APC in a context that isrecognized by a T cell by quantifying the density of epitope-HLA class Icomplexes on the cell surface. Quantitation can be performed by directlymeasuring the amount of peptide eluted from the APC (see, e.g., Sijts etal., J. Immunol. 156:683-692, 1996; Demotz et al., Nature 342:682-684,1989); or the number of peptide-HLA class I complexes can be estimatedby measuring the amount of lysis or lympholine release induced bydiseased or transfected target cells, and then determining theconcentration of peptide necessary to obtain equivalent levels of lysisor lympholine release (see, e.g., Kageyama et al., J. Immunol.154:567-576, 1995).

Alternatively, immunogenicity is confirmed through in vivo injectionsinto mice and subsequent in vitro assessment of CTL and HTL activity,which are analyzed using cytotoxicity and proliferation assays,respectively, as detailed e.g., in Alexander et al., Immunity 1:751-761,1994.

For example, to confirm the capacity of a DNA minigene constructcontaining at least one HLA-A2 supermotif peptide to induce CTLs invivo, HLA-A_(2.1)/K^(b) transgenic mice, for example, are immunizedintramuscularly with 100 μg of naked cDNA. As a means of comparing thelevel of CTLs induced by cDNA immunization, a control group of animalsis also immunized with an actual peptide composition that comprisesmultiple epitopes synthesized as a single polypeptide as they would beencoded by the minigene.

Splenocytes from immunized animals are stimulated twice with each of therespective compositions (peptide epitopes encoded in the minigene or thepolyepitopic peptide), then assayed for peptide-specific cytotoxicactivity in a ⁵¹Cr release assay. The results indicate the magnitude ofthe CTL response directed against the A2-restricted epitope, thusindicating the in vivo immunogenicity of the minigene vaccine andpolyepitopic vaccine.

It is, therefore, found that the minigene elicits immune responsesdirected toward the HLA-A2 supermotif peptide epitopes as does thepolyepitopic peptide vaccine. A similar analysis is also performed usingother HLA-A3 and HLA-B7 transgenic mouse models to assess CTL inductionby HLA-A3 and HLA-B7 motif or supermotif epitopes, whereby it is alsofound that the minigene elicits appropriate immune responses directedtoward the provided epitopes.

To confirm the capacity of a class II epitope-encoding minigene toinduce HTLs in vivo, DR transgenic mice, or for those epitopes thatcross react with the appropriate mouse MHC molecule, I-A^(b)-restrictedmice, for example, are immunized intramuscularly with 100 μg of plasmidDNA. As a means of comparing the level of HTLs induced by DNAimmunization, a group of control animals is also immunized with anactual peptide composition emulsified in complete Freund's adjuvant CD4+T cells, i.e. HTLs, are purified from splenocytes of immunized animalsand stimulated with each of the respective compositions (peptidesencoded in the minigene). The HTL response is measured using a³H-thymidine incorporation proliferation assay, (see, e.g., Alexander etal. Immunity 1:751-761, 1994). The results indicate the magnitude of theHTL response, thus demonstrating the in vivo immunogenicity of theminigene.

DNA minigenes, constructed as described in the previous Example, canalso be confirmed as a vaccine in combination with a boosting agentusing a prime boost protocol. The boosting agent can consist ofrecombinant protein (e.g., Barnett et al., Aids Res. and HumanRetroviruses 14, Supplement 3:S299-S309, 1998) or recombinant vaccinia,for example, expressing a minigene or DNA encoding the complete proteinof interest (see, e.g., Hanke et al., Vaccine 16:439-445, 1998; Sedegahet al., Proc. Natl. Acad. Sci. USA 95:7648-53, 1998; Hanke andMcMichael, Immunol. Letters 66:177-181, 1999; and Robinson et al.,Nature Med. 5:526-34, 1999).

For example, the efficacy of the DNA minigene used in a prime boostprotocol is initially evaluated in transgenic mice. In this example,A2.1/K^(b) transgenic mice are immunized IM with 100 μg of a DNAminigene encoding the immunogenic peptides including at least one HLA-A2supermotif-bearing peptide. After an incubation period (ranging from 3-9weeks), the mice are boosted IP with 10⁷ pfu/mouse of a recombinantvaccinia virus expressing the same sequence encoded by the DNA minigene.Control mice are immunized with 100 μg of DNA or recombinant vacciniawithout the minigene sequence, or with DNA encoding the minigene, butwithout the vaccinia boost. After an additional incubation period of twoweeks, splenocytes from the mice are immediately assayed forpeptide-specific activity in an ELISPOT assay. Additionally, splenocytesare stimulated in vitro with the A2-restricted peptide epitopes encodedin the minigene and recombinant vaccinia, then assayed forpeptide-specific activity in an alpha, beta and/or gamma IFN ELISA.

It is found that the minigene utilized in a prime-boost protocol elicitsgreater immune responses toward the HLA-A2 supermotif peptides than withDNA alone. Such an analysis can also be performed using HLA-A11 orHLA-B7 transgenic mouse models to assess CTL induction by HLA-A3 orHLA-B7 motif or supermotif epitopes. The use of prime boost protocols inhumans is described below in the Example entitled “Induction of CTLResponses Using a Prime Boost Protocol.”

Example 22 Peptide Composition for Prophylactic Uses

Vaccine compositions of the present invention can be used to prevent205P1B5 expression in persons who are at risk for tumors that bear thisantigen. For example, a polyepitopic peptide epitope composition (or anucleic acid comprising the same) containing multiple CTL and HTLepitopes such as those selected in the above Examples, which are alsoselected to target greater than 80% of the population, is administeredto individuals at risk for a 205P1B5-associated tumor.

For example, a peptide-based composition is provided as a singlepolypeptide that encompasses multiple epitopes. The vaccine is typicallyadministered in a physiological solution that comprises an adjuvant,such as Incomplete Freunds Adjuvant. The dose of peptide for the initialimmunization is from about 1 to about 50,000 μg, generally 100-5,000 μg,for a 70 kg patient. The initial administration of vaccine is followedby booster dosages at 4 weeks followed by evaluation of the magnitude ofthe immune response in the patient, by techniques that determine thepresence of epitope-specific CTL populations in a PBMC sample.Additional booster doses are administered as required. The compositionis found to be both safe and efficacious as a prophylaxis against205P1B5-associated disease.

Alternatively, a composition typically comprising transfecting agents isused for the administration of a nucleic acid-based vaccine inaccordance with methodologies known in the art and disclosed herein.

Example 23 Polyepitopic Vaccine Compositions Derived from Native 205P1B5Sequences

A native 205P1B5 polyprotein sequence is analyzed, preferably usingcomputer algorithms defined for each class I and/or class II supermotifor motif, to identify “relatively short” regions of the polyprotein thatcomprise multiple epitopes. The “relatively short” regions arepreferably less in length than an entire native antigen. This relativelyshort sequence that contains multiple distinct or overlapping, “nested”epitopes is selected; it can be used to generate a minigene constructThe construct is engineered to express the peptide, which corresponds tothe native protein sequence. The “relatively short” peptide is generallyless than 250 amino acids in length, often less than 100 amino acids inlength, preferably less than 75 amino acids in length, and morepreferably less than 50 amino acids in length. The protein sequence ofthe vaccine composition is selected because it has maximal number ofepitopes contained within the sequence, i.e., it has a highconcentration of epitopes. As noted herein, epitope motifs may be nestedor overlapping (i.e., frame shifted relative to one another). Forexample, with overlapping epitopes, two 9-mer epitopes and one 10 merepitope can be present in a 10 amino acid peptide. Such a vaccinecomposition is administered for therapeutic or prophylactic purposes.

The vaccine composition will include, for example, multiple CTL epitopesfrom 205P1B5 antigen and at least one HTL epitope. This polyepitopicnative sequence is administered either as a peptide or as a nucleic acidsequence which encodes the peptide. Alternatively, an analog can be madeof this native sequence, whereby one or more of the epitopes comprisesubstitutions that alter the cross-reactivity and/or binding affinityproperties of the polyepitopic peptide.

The embodiment of this example provides for the possibility that an asyet undiscovered aspect of immune system processing will apply to thenative nested sequence and thereby facilitate the production oftherapeutic or prophylactic immune response-inducing vaccinecompositions. Additionally such an embodiment provides for thepossibility of motif-bearing epitopes for an HLA makeup that ispresently unknown. Furthermore, this embodiment (excluding an analogedembodiment) directs the immune response to multiple peptide sequencesthat are actually present in native 205P1B5, thus avoiding the need toevaluate any junctional epitopes. Lastly, the embodiment provides aneconomy of scale when producing peptide or nucleic acid vaccinecompositions.

Related to this embodiment, computer programs are available in the artwhich can be used to identify in a target sequence, the greatest numberof epitopes per sequence length.

Example 24 Polyepitopic Vaccine Compositions from Multiple Antigens

The 205P1B5 peptide epitopes of the present invention are used inconjunction with epitopes from other target tumor-associated antigens,to create a vaccine composition that is useful for the prevention ortreatment of cancer that expresses 205P1B5 and such other antigens. Forexample, a vaccine composition can be provided as a single polypeptidethat incorporates multiple epitopes from 205P1B5 as well astumor-associated antigens that are often expressed with a target cancerassociated with 205P1B5 expression, or can be administered as acomposition comprising a cocktail of one or more discrete epitopes.Alternatively, the vaccine can be administered as a minigene constructor as dendritic cells which have been loaded with the peptide epitopesin vitro.

Example 25 Use of Peptides to Evaluate an Immune Response

Peptides of the invention may be used to analyze an immune response forthe presence of specific antibodies, CTL or HTL directed to 205P1B5.Such an analysis can be performed in a manner described by Ogg et al.,Science 279:2103-2106, 1998. In this Example, peptides in accordancewith the invention are used as a reagent for diagnostic or prognosticpurposes, not as an immunogen.

In this example highly sensitive human leukocyte antigen tetramericcomplexes (“tetrames”) are used for a cross-sectional analysis of, forexample, 205P1B5 HLA-A*0201-specific CTL frequencies from HLAA*0201-positive individuals at different stages of disease or followingimmunization comprising an 205P1B5 peptide containing an A*0201 motif.Tetrameric complexes are synthesized as described (Musey et al., N.Engl. J. Med. 337:1267, 1997). Briefly, purified HLA heavy chain (A*0201in this example) and β2-microglobulin are synthesized by means of aprokaryotic expression system. The heavy chain is modified by deletionof the transmembrane-cytosolic tail and COOH-terminal addition of asequence containing a BirA enzymatic biotinylation site. The heavychain, β2-microglobulin, and peptide are refolded by dilution. The 45-kDrefolded product is isolated by fast protein liquid chromatography andthen biotinylated by BirA in the presence of biotin (Sigma, St. Louis,Mo.), adenosine 5′ triphosphate and magnesium.Streptavidin-phycoerythrin conjugate is added in a 1:4 molar ratio, andthe tetrameric product is concentrated to 1 mg/ml. The resulting productis referred to as tetramer-phycoerythrin.

For the analysis of patient blood samples, approximately one millionPBMCs are centrifuged at 300 g for 5 minutes and resuspended in 50 μl ofcold phosphate-buffered saline. Tri-color analysis is performed with thetetramer-phycoerythrin, along with anti-CD8-Tricolor, and anti-CD38. ThePBMCs are incubated with tetramer and antibodies on ice for 30 to 60 minand then washed twice before formaldehyde fixation. Gates are applied tocontain>99.98% of control samples. Controls for the tetramers includeboth A*0201-negative individuals and A*0201-positive non-diseaseddonors. The percentage of cells stained with the tetramer is thendetermined by flow cytometry. The results indicate the number of cellsin the PBMC sample that contain epitope-restricted CTLs, thereby readilyindicating the extent of immune response to the 205P1B5 epitope, andthus the status of exposure to 205P1B5, or exposure to a vaccine thatelicits a protective or therapeutic response.

Example 26 Use of Peptide Epitopes to Evaluate Recall Responses

The peptide epitopes of the invention are used as reagents to evaluate Tcell responses, such as acute or recall responses, in patients. Such ananalysis may be performed on patients who have recovered from205P1B5-associated disease or who have been vaccinated with an 205P1B5vaccine.

For example, the class I restricted CTL response of persons who havebeen vaccinated may be analyzed. The vaccine may be any 205P1B5 vaccine.PBMC are collected from vaccinated individuals and HLA typed.Appropriate peptide epitopes of the invention that, optimally, bearsupermotifs to provide cross-reactivity with multiple HLA supertypefamily members, are then used for analysis of samples derived fromindividuals who bear that HLA type.

PBMC from vaccinated individuals are separated on Ficoll-Histopaquedensity gradients (Sigma Chemical Co., St. Louis, Mo.), washed threetimes in HBSS (GIBCO Laboratories), resuspended in RPMI-1640 (GIBCOLaboratories) supplemented with L-glutamine (2 nM,) penicillin (50μ/ml), streptomycin (50 μg/ml), and Hepes (10 mM) containing 10%heat-inactivated human AB serum (complete RPMI) and plated usingmicroculture formats. A synthetic peptide comprising an epitope of theinvention is added at 10 μg/ml to each well and HBV core 128-140 epitopeis added at 1 μg/ml to each well as a source of T cell help during thefirst week of stimulation.

In the microculture format, 4×10⁵ PBMC are stimulated with peptide in 8replicate cultures in 96-well round bottom plate in 100 μl/well ofcomplete RPMI. On days 3 and 10, 100 μl of complete RPMI and 20 U/mlfinal concentration of rIL-2 are added to each well. On day 7 thecultures are transferred into a 96-well flat-bottom plate andrestimulated with peptide, rIL-2 and 10⁵ irradiated (3,000 rad)autologous feeder cells. The cultures are tested for cytotoxic activityon day 14. A positive CTL response requires two or more of the eightreplicate cultures to display greater than 10% specific ⁵¹Cr release,based on comparison with non-diseased control subjects as previouslydescribed (Rehermann, et al., Nature Med. 2:1104,1108, 1996; Rehermannet al., J. Clin. Invest. 97:1655-1665, 1996; and Rehermann et al. J.Clin. Invest. 98:1432-1440, 1996).

Target cell lines are autologous and allogeneic EBV-transformed B-LCLthat are either purchased from the American Society forHistocompatibility and Immunogenetics (ASHI, Boston, Mass.) orestablished from the pool of patients as described (Guilhot, et al. J.Virol. 66:2670-2678, 1992).

Cytotoxicity assays are performed in the following manner. Target cellsconsist of either allogeneic HLA-matched or autologous EBV-transformed Blymphoblastoid cell lane that are incubated overnight with the syntheticpeptide epitope of the invention at 10 μM, and labeled with 100 μCi of⁵¹Cr (Amersham Corp., Arlington Heights, Ill.) for 1 hour after whichthey are washed four times with HBSS.

Cytolytic activity is determined in a standard 4-h, split well ⁵¹Crrelease assay using U-bottomed 96 well plates containing 3,000targets/well. Stimulated PBMC are tested at effector/target (E/T) ratiosof 20-50:1 on day 14. Percent cytotoxicity is determined from theformula: 100×[(experimental release−spontaneous release)/maximumrelease−spontaneous release)]. Maximum release is determined by lysis oftargets by detergent (2% Triton X-100; Sigma Chemical Co., St. Louis,Mo.). Spontaneous release is<25% of maximum release for all experiments.

The results of such an analysis indicate the extent to whichHLA-restricted CTL populations have been stimulated by previous exposureto 205P1B5 or an 205P1B5 vaccine.

Similarly, Class II restricted HTL responses may also be analyzed.Purified PBMC are cultured in a 96-well flat bottom plate at a densityof 1.5×10⁵ cells/well and are stimulated with 10 μg/ml synthetic peptideof the invention, whole 205P1B5 antigen, or PHA. Cells are routinelyplated in replicates of 4-6 wells for each condition. After seven daysof culture, the medium is removed and replaced with fresh mediumcontaining 10 U/ml IL-2. Two days later, 1 μCi ³H-thymidine is added toeach well and incubation is continued for an additional 18 hours.Cellular DNA is then harvested on glass fiber mats and analyzed for³H-thymidine incorporation. Antigen-specific T cell proliferation iscalculated as the ratio of ³H-thymidine incorporation in the presence ofantigen divided by the ³H-thymidine incorporation in the absence ofantigen.

Example 27 Induction of Specific CTL Response In Humans

A human clinical trial for an immunogenic composition comprising CTL andHTL epitopes of the invention is set up as an IND Phase I, doseescalation study and carried out as a randomized, double-blind,placebo-controlled trial. Such a trial is designed, for example, asfollows:

A total of about 27 individuals are enrolled and divided into 3 groups:

Group I: 3 subjects are injected with placebo and 6 subjects areinjected with 5 μg of peptide composition;

Group II: 3 subjects are injected with placebo and 6 subjects areinjected with 50 μg peptide composition;

Group III: 3 subjects are injected with placebo and 6 subjects areinjected with 500 μg of peptide composition.

After 4 weeks following the first injection, all subjects receive abooster inoculation at the same dosage.

The endpoints measured in this study relate to the safety andtolerability of the peptide composition as well as its immunogenicity.Cellular immune responses to the peptide composition are an index of theintrinsic activity of this the peptide composition, and can therefore beviewed as a measure of biological efficacy. The following summarize theclinical and laboratory data that relate to safety and efficacyendpoints.

Safety:

The incidence of adverse events is monitored in the placebo and drugtreatment group and assessed in terms of degree and reversibility.

Evaluation of Vaccine Efficacy:

For evaluation of vaccine efficacy, subjects are bled before and afterinjection. Peripheral blood mononuclear cells are isolated from freshheparinized blood by Ficoll-Hypaque density gradient centrifugation,aliquoted in freezing media and stored frozen. Samples are assayed forCTL and HTL activity.

The vaccine is found to be both safe and efficacious.

Example 28 Phase II Trials in Patients Expressing 205P1B5

Phase II trials are performed to study the effect of administering theCTL-HTL peptide compositions to patients having cancer that expresses205P1B5. The main objectives of the trial are to determine an effectivedose and regimen for inducing CTLs in cancer patients that express205P1B5, to establish the safety of inducing a CTL and HTL response inthese patients, and to see to what extent activation of CTLs improvesthe clinical picture of these patients, as manifested, e.g., by thereduction and/or shrinking of lesions. Such a study is designed, forexample, as follows:

The studies are performed in multiple centers. The trial design is anopen-label, uncontrolled, dose escalation protocol wherein the peptidecomposition is administered as a single dose followed six weeks later bya single booster shot of the same dose. The dosages are 50, 500 and5,000 micrograms per injection. Drug-associated adverse effects(severity and reversibility) are recorded.

There are three patient groupings. The first group is injected with 50micrograms of the peptide composition and the second and third groupswith 500 and 5,000 micrograms of peptide composition, respectively. Thepatients within each group range in age from 21-65 and represent diverseethnic backgrounds. All of them have a tumor that expresses 205P1B5.

Clinical manifestations or antigen-specific T-cell responses aremonitored to assess the effects of administering the peptidecompositions. The vaccine composition is found to be both safe andefficacious in the treatment of 205P1B5-associated disease.

Example 29 Induction of CTL Responses Using a Prime Boost Protocol

A prime boost protocol similar in its underlying principle to that usedto confirm the efficacy of a DNA vaccine in transgenic mice, such asdescribed above in the Example entitled “The Plasmid Construct and theDegree to Which It Induces Immunogenicity,” can also be used for theadministration of the vaccine to humans. Such a vaccine regimen caninclude an initial administration of, for example, naked DNA followed bya boost using recombinant virus encoding the vaccine, or recombinantprotein/polypeptide or a peptide mixture administered in an adjuvant.

For example, the initial immunization may be performed using anexpression vector, such as that constructed in the Example entitled“Construction of ‘Minigene’ Multi-Epitope DNA Plasmids” in the form ofnaked nucleic acid administered IM (or SC or ID) in the amounts of 0.5-5mg at multiple sites. The nucleic acid (0.1 to 1000 μg) can also beadministered using a gene gun. Following an incubation period of 3-4weeks, a booster dose is then administered. The booster can berecombinant fowlpox virus administered at a dose of 5−10⁷ to 5×10⁹ pfu.An alternative recombinant virus, such as an MVA, canarypox, adenovirus,or adeno-associated virus, can also be used for the booster, or thepolyepitopic protein or a mixture of the peptides can be administered.For evaluation of vaccine efficacy, patient blood samples are obtainedbefore immunization as well as at intervals following administration ofthe initial vaccine and booster doses of the vaccine. Peripheral bloodmononuclear cells are isolated from fresh heparinized blood byFicoll-Hypaque density gradient centrifugation, aliquoted in freezingmedia and stored frozen. Samples are assayed for CTL and HTL activity.

Analysis of the results indicates that a magnitude of responsesufficient to achieve a therapeutic or protective immunity against205P1B5 is generated.

Example 30 Administration of Vaccine Compositions Using Dendritic Cells(DC)

Vaccines comprising peptide epitopes of the invention can beadministered using APCs, or “professional” APCs such as DC. In thisexample, peptide-pulsed DC are administered to a patient to stimulate aCTL response in vivo. In this method, dendritic cells are isolated,expanded, and pulsed with a vaccine comprising peptide CTL and HTLepitopes of the invention. The dendritic cells are infused back into thepatient to elicit CTL and HTL responses in vivo. The induced CTL and HTLthen destroy or facilitate destruction, respectively, of the targetcells that bear the 205P1B5 protein from which the epitopes in thevaccine are derived.

For example, a cocktail of epitope-comprising peptides is administeredex vivo to PBMC, or isolated DC therefrom. A pharmaceutical tofacilitate harvesting of DC can be used, such as Progenipoietin™(Monsanto, St. Louis, Mo.) or GM-CSF/IL-4. After pulsing the DC withpeptides, and prior to reinfusion into patients, the DC are washed toremove unbound peptides.

As appreciated clinically, and readily determined by one of skill basedon clinical outcomes, the number of DC reinfused into the patient canvary (see, e.g., Nature Med. 4:328, 1998; Nature Med. 2:52, 1996 andProstate 32:272, 1997). Although 2−50×10⁶ DC per patient are typicallyadministered, larger number of DC, such as 10⁷ or 10⁸ can also beprovided. Such cell populations typically contain between 50-90% DC.

In some embodiments, peptide-loaded PBMC are injected into patientswithout purification of the DC. For example, PBMC generated aftertreatment with an agent such as Progenipoietin™ are injected intopatients without purification of the DC. The total number of PBMC thatare administered often ranges from 10⁸ to 10¹⁰. Generally, the celldoses injected into patients is based on the percentage of DC in theblood of each patient, as determined, for example, by immunofluorescenceanalysis with specific anti-DC antibodies. Thus, for example, ifProgenipoietin™ mobilizes 2% DC in the peripheral blood of a givenpatient, and that patient is to receive 5×10⁶ DC, then the patient willbe injected with a total of 2.5×10⁸ peptide-loaded PBMC. The percent DCmobilized by an agent such as Progenipoietin™ is typically estimated tobe between 2-10%, but can vary as appreciated by one of skill in theart.

Ex Vivo Activation of CTL/HTL Responses

Alternatively, ex vivo CTL or HTL responses to 205P1B5 antigens can beinduced by incubating, in tissue culture, the patient's, or geneticallycompatible, CTL or HTL precursor cells together with a source of APC,such as DC, and immunogenic peptides. After an appropriate incubationtime (typically about 7-28 days), in which the precursor cells areactivated and expanded into effector cells, the cells are infused intothe patient, where they will destroy (CTL) or facilitate destruction(HTL) of their specific target cells, i.e., tumor cells.

Example 31 An Alternative Method of Identifying and ConfirmingMotif-Bearing Peptides

Another method of identifying and confirming motif-bearing peptides isto elute them from cells bearing defined MHC molecules. For example, EBVtransformed B cell lines used for tissue typing have been extensivelycharacterized to determine which HLA molecules they express. la certaincases these cells express only a single type of HLA molecule. Thesecells can be transfected with nucleic acids that express the antigen ofinterest, e.g. 205P1B5. Peptides produced by endogenous antigenprocessing of peptides produced as a result of transfection will thenbind to HLA molecules within the cell and be transported and displayedon the cell's surface. Peptides are then eluted from the HLA moleculesby exposure to mild acid conditions and their amino acid sequencedetermined, e.g., by mass spectral analysis (e.g., Kubo et al., J.Immunol. 352:3913, 1994). Because the majority of peptides that bind aparticular HLA molecule are motif-bearing, this is an alternativemodality for obtaining the motif-bearing peptides correlated with theparticular HLA molecule expressed on the cell.

Alternatively, cell lines that do not express endogenous HLA moleculescan be transfected with an expression construct encoding a single HLAallele. These cells can then be used as described, i.e., they can thenbe transfected with nucleic acids that encode 205P1B5 to isolatepeptides corresponding to 205P1B5 that have been presented on the cellsurface. Peptides obtained from such an analysis will bear motif(s) thatcorrespond to binding to the single HLA allele that is expressed in thecell.

As appreciated by one in the art, one can perform a similar analysis ona cell bearing more than one HLA allele and subsequently determinepeptides specific for each HLA allele expressed. Moreover, one of skillwould also recognize that means other than transfection, such as loadingwith a protein antigen, can be used to provide a source of antigen tothe cell.

Example 32 Complementary Polynucleotides

Sequences complementary to the 205P1B5-encoding sequences, or any partsthereof, are used to detect, decrease, or inhibit expression ofnaturally occurring 205P1B5. Although use of oligonucleotides comprisingfrom about 15 to 30 base pairs is described, essentially the sameprocedure is used with smaller or with larger sequence fragments.Appropriate oligonucleotides are designed using, e.g., OLIGO 4.06software (National Biosciences) and the coding sequence of 205P1B5. Toinhibit transcription, a complementary oligonucleotide is designed fromthe most unique 5′ sequence and used to prevent promoter binding to thecoding sequence. To inhibit translation, a complementary oligonucleotideis designed to prevent ribosomal binding to the 205P1B5-encodingtranscript.

Example 33 Purification of Naturally-Occurring or Recombinant 205P1B5Using 205P1B5 Specific Antibodies

Naturally occurring or recombinant 205P1B5 is substantially purified byimmunoaffinity chromatography using antibodies specific for 205P1B5. Animmunoaffinity column is constructed by covalently coupling anti-205P1B5antibody to an activated chromatographic resin, such as CNBr-activatedSEPHAROSE (Amersham Pharmacia Biotech). After the coupling, the resin isblocked and washed according to the manufacturer's instructions.

Media containing 205P1B5 are passed over the immunoaffinity column, andthe column is washed under conditions that allow the preferentialabsorbance of 205P1B5 (e.g., high ionic strength buffers in the presenceof detergent). The column is eluted under conditions that disruptantibody/205P1B5 binding (e.g., a buffer of pH 2 to pH 3, or a highconcentration of a chaotrope, such as urea or thiocyanate ion), and GCRPis collected.

Example 34 Identification of Molecules which Interact with 205P1B5

205P1B5, or biologically active fragments thereof, are labeled with 1211 Bolton-Hunter reagent. (See, e.g., Bolton et al. (1973) Biochem. J.133:529.) Candidate molecules previously arrayed in the wells of amulti-well plate are incubated with the labeled 205P1B5, washed, and anywells with labeled 205P1B5 complex are assayed. Data obtained usingdifferent concentrations of 205P1B5 are used to calculate values for thenumber, affinity, and association of 205P1B5 with the candidatemolecules.

Example 35 In Vivo Assay for 205P1B5 Tumor Growth Promotion

The effect of the 205P1B5 protein on tumor cell growth is evaluated invivo by gene overexpression in tumor-bearing mice. For example, SCIDmice are injected subcutaneously on each flank with 1×10⁶ of either PC3,TSUPR1, or DU145 cells containing tkNeo empty vector or 205P1B5. Atleast two strategies may be used: (1) Constitutive 205P1B5 expressionunder regulation of a promoter such as a constitutive promoter obtainedfrom the genomes of viruses such as polyoma virus, fowlpox virus (UK2,211,504 published 5 Jul. 1989), adenovirus (such as Adenovirus 2),bovine papilloma virus, avian sarcoma virus, cytomegalovirus, aretrovirus, hepatitis-B virus and Simian Virus 40 (SV40), or fromheterologous mammalian promoters, e.g., the actin promoter or animmunoglobulin promoter, provided such promoters are compatible with thehost cell systems, and (2) Regulated expression under control of aninducible vector system, such as ecdysone, tet, etc., provided suchpromoters are compatible with the host cell systems. Tumor volume isthen monitored at the appearance of palpable tumors and followed overtime to determine if 205P1B5-expressing cells grow at a faster rate andwhether tumors produced by 205P1B5-expressing cells demonstratecharacteristics of altered aggressiveness (e.g. enhanced metastasis,vascularization, reduced responsiveness to chemotherapeutic drugs).

Additionally, mice can be implanted with 1×10⁵ of the same cellsorthotopically to determine if 205P1B5 has an effect on local growth inthe prostate or on the ability of the cells to metastasize, specificallyto lungs, lymph nodes, and bone marrow.

The assay is also useful to determine the 205P1B5 inhibitory effect ofcandidate therapeutic compositions, such as for example, 205P1B5intrabodies, 205P1B5 antisense molecules and ribozymes.

Example 36 205P1B5 Monoclonal Antibody-Mediated Inhibition of ProstateTumors In Vivo

The significant expression of 205P1B5, in cancer tissues, together withits restrictive expression in normal tissues along with its expectedcell surface expression makes 205P1B5 an excellent target for antibodytherapy. Similarly, 205P1B5 is a target for T cell-based immunotherapy.Thus, the therapeutic efficacy of anti-205P1B5 mAbs in human prostatecancer xenograft mouse models is evaluated by using androgen-independentLAPC4 and LAPC-9 xenograft (Craft, N., et al., Cancer Res, 1999. 59(19):p. 5030-6) and the androgen independent recombinant cell linePC3-205P1B5 (see, e.g., Kaighn, M E., et al., Invest. Urol, 1979. 17(1):p. 16-23).

Antibody efficacy on tumor growth and metastasis formation is studied,e.g., in a mouse orthotopic prostate cancer xenograft model. Theantibodies can be unconjugated, as discussed in this Example, or can beconjugated to a therapeutic modality, as appreciated in the art.Anti-205P1B5 mAbs inhibit formation of both the androgen-dependentLAPC-9 and androgen-independent PC3-205P1B5 tumor xenografts.Anti-205P1B5 mAbs also retard the growth of established orthotopictumors and prolonged survival of tumor-bearing mice. These resultsindicate the utility of anti-205P1B5 mAbs in the treatment of local andadvanced stages of prostate cancer. (See, e.g., (Saffran, D., et al.,PNAS 10:1073-1078 or world wide web URLpnas.org/cgi/doi/10.1073/pnas.051624698)

Administration of the anti-205P1B5 mAbs led to retardation ofestablished orthotopic tumor growth and inhibition of metastasis todistant sites, resulting in a significant prolongation in the survivalof tumor-bearing mice. These studies indicate that 205P1B5 as anattractive target for immunotherapy and demonstrate the therapeuticpotential of anti-205P1B5 mAbs for the treatment of local and metastaticprostate cancer. This example demonstrates that unconjugated 205P1B5monoclonal antibodies are effective to inhibit the growth of humanprostate tumor xenografts grown in SCID mice; accordingly a combinationof such efficacious monoclonal antibodies is also effective.

Tumor Inhibition Using Multiple Unconjugated 205P1B5 mAbs

Materials and Methods

205P1B5 Monoclonal Antibodies:

Monoclonal antibodies are raised against 205P1B5 as described in theExample entitled “Generation of 205P1B5 Monoclonal Antibodies (mAbs).”The antibodies are characterized by ELISA, Western blot, FACS, andimmunoprecipitation for their capacity to bind 205P1B5. Epitope mappingdata for the anti-205P1B5 mAbs, as determined by ELISA and Westernanalysis, recognize epitopes on the 205P1B5 protein. Immunohistochemicalanalysis of prostate cancer tissues and cells with these antibodies isperformed.

The monoclonal antibodies are purified from ascites or hybridoma tissueculture supernatants by Protein-G Sepharose chromatography, dialyzedagainst PBS, filter sterilized, and stored at −20° C. Proteindeterminations are performed by a Bradford assay (Bio-Rad, Hercules,Calif.). A therapeutic monoclonal antibody or a cocktail comprising amixture of individual monoclonal antibodies is prepared and used for thetreatment of mice receiving subcutaneous or orthotopic injections ofLAPC-9 prostate tumor xenografts.

Prostate Cancer Xenografts and Cell Lines

The LAPC-9 xenograft, which expresses a wild-type androgen receptor andproduces prostate-specific antigen (PSA), is passaged in 6- to8-week-old male ICR-severe combined immunodeficient (SCID) mice (TaconicFarms) by s.c. trocar implant (Craft, N., et al., supra). Single-cellsuspensions of LAPC-9 tumor cells are prepared as described in Craft, etal. The prostate carcinoma cell line PC3 (American Type CultureCollection) is maintained in DMEM supplemented with L-glutamine and 10%(vol/vol) FBS.

A PC3-205P1B5 cell population is generated by retroviral gene transferas described in Hubert, R S., et al., STEAP: a prostate-specificcell-surface antigen highly expressed in human prostate tumors. ProcNatl Acad Sci USA, 1999. 96(25): p. 14523-8. Anti-205P1B5 staining isdetected by using an FITC-conjugated goat anti-mouse antibody (SouthernBiotechnology Associates) followed by analysis on a Coulter Epics-XL flow cytometer.

Xenograft Mouse Models.

Subcutaneous (s.c.) tumors are generated by injection of 1×10⁶ LAPC-9,PC3, or PC3-205P1B5 cells mixed at a 1:1 dilution with Matrigel(Collaborative Research) in the right flank of male SCID mice. To testantibody efficacy on tumor formation, i.p. antibody injections arestarted on the same day as tumor-cell injections. As a control, mice areinjected with either purified mouse IgG (ICN) or PBS; or a purifiedmonoclonal antibody that recognizes an irrelevant antigen not expressedin human cells. In preliminary studies, no difference is found betweenmouse IgG or PBS on tumor growth. Tumor sizes are determined by verniercaliper measurements, and the tumor volume is calculated aslength×width×height Mice with s.c. tumors greater than 1.5 cm indiameter are sacrificed. PSA levels are determined by using a PSA ELISAkit (Anogen, Mississauga, Ontario). Circulating levels of anti-205P1B5mAbs are determined by a capture ELISA kit (Bethyl Laboratories,Montgomery, Tex.). (See, e.g., (Saffran, D., et al., PNAS 10: 1073-1078or world wide web URL pnas.org/cgi/doi/10.1073/pnas.051624698)

Orthotopic injections are performed under anesthesia by usingketamine/xylazine. An incision is made through the abdominal muscles toexpose the bladder and seminal vesicles, which then are deliveredthrough the incision to expose the dorsal prostate. LAPC-9 cells (5×10⁵)mixed with Matrigel are injected into each dorsal lobe in a 10-μlvolume. To monitor tumor growth, mice are bled on a weekly basis fordetermination of PSA levels. Based on the PSA levels, the mice aresegregated into groups for the appropriate treatments. To test theeffect of anti-205P1B5 mAbs on established orthotopic tumors, i.p.antibody injections are started when PSA levels reach 2-80 ng/ml.

Anti-205P1B5 mAbs Inhibit Growth of 205P1B5-Expressing Prostate-CancerTumors

The effect of anti-205P1B5 mAbs on tumor formation is tested by usingthe LAPC-9 orthotopic model. As compared with the s.c. tumor model, theorthotopic model, which requires injection of tumor cells directly inthe mouse prostate, results in a local tumor growth, development ofmetastasis in distal sites, deterioration of mouse health, andsubsequent death (Saffran, D., et al., PNAS supra; Fu, X., et al., IntJ. Cancer, 1992. 52(6): p. 987-90; Kubota, T., J. Cell Biochem, 1994.56(1): p. 4-8). The features make the orthotopic model morerepresentative of human disease progression and allowed us to follow thetherapeutic effect of mAbs on clinically relevant end points.

Accordingly, LAPC-9 tumor cells are injected into the mouse prostate,and 2 days later, the mice are segregated into two groups and treatedwith either: a) 50-2000 μg, usually 200-500 μg, of anti-205P1B5 Ab, orb) PBS three times per week for two to five weeks. Mice are monitoredweekly for circulating PSA levels as an indicator of tumor growth.

A major advantage of the orthotopic prostate-cancer model is the abilityto study the development of metastases. Formation of metastasis in micebearing established orthotopic tumors is studies by IHC analysis on lungsections using an antibody against a prostate-specific cell-surfaceprotein STEAP expressed at high levels in LAPC-9 xenografts (Hubert, R.S., et al., Proc Natl Acad Sci USA, 1999. 96(25): p. 14523-8).

Mice bearing established orthotopic LAPC-9 tumors are administered 1000μg injections of either anti-205P1B5 mAb or PBS over a 4-week period.Mice in both groups are allowed to establish a high tumor burden (PSAlevels greater than 300 ng/ml), to ensure a high frequency of metastasisformation in mouse lungs. Mice then are killed and their prostate andlungs are analyzed for the presence of LAPC-9 cells by anti-STEAP IHCanalysis.

These studies demonstrate a broad anti-tumor efficacy of anti-205P1B5antibodies on initiation and progression of prostate cancer in xenograftmouse models. Anti-205P1B5 antibodies inhibit tumor formation of bothandrogen-dependent and androgen-independent tumors as well as retardingthe growth of already established tumors and prolong the survival oftreated mice. Moreover, anti-205P1B5 mAbs demonstrate a dramaticinhibitory effect on the spread of local prostate tumor to distal sites,even in the presence of a large tumor burden. Thus, anti-205P1B5 mAbsare efficacious on major clinically relevant end points/PSA levels(tumor growth), prolongation of survival, and health.

Example 37 Comparison of 205P1B5 to Known Genes

205P1B5 is a 529 amino acid protein with a calculated MW of 59.7 kDa,and pI of 5.69. As shown in FIG. 4, 205P1B5 shows 100% identity to thehuman cholinergic receptor, nicotinic, alpha polypeptide 2(gi:12734121). 205P1B5 is predicted to be a cell surface protein thatfunctions as an ion transporter (Vizi E S and Lendvai B. Brain Res BrainRes Rev. 1999, 30:219; Shao Z and Yakel J. L. J. Physiol. 2000,527:507). As described by Vizi et Lendvai nicotinic acetylcholinereceptors participates in calcium and sodium signaling in both synapticand non-synaptic locations (Vizi E S and Lendvai B. Brain Res Brain ResRev. 1999, 30:219). The expression of nicotinic cholinergic receptorshas been documented in small cell lung cancer, where they arefunctionally active and induce calcium flux in response to stimuli(Codignola A et al., FEBS Lett. 1994, 342:286). Thus, substances thatmodulate the presence or effect of cholinergic receptors are used fordiagnosis, prophylaxis, prognosis and/or treatment of a diseasecondition disclosed herein, such as a cancer listed in Table I.

Example 38 Identification of Potential Signal Transduction Pathways

Many mammalian proteins have been reported to interact with signalingmolecules and to participate in, regulating signaling pathways (JNeurochem. 2001; 76:217-223). Using immunoprecipitation and Westernblotting techniques, proteins are identified that associate with 205P1B5and mediate signaling events. Several pathways known to play a role incancer biology can be regulated by several of these genes, includingphospholipid pathways such as P13K, AKT, etc., adhesion and migrationpathways, including FAK, Rho, Rac-1, etc., as well as mitogenic/survivalcascades such as ERK, p38, etc. (Cell Growth Differ. 2000,11:279; J.Biol. Chem. 1999,274:801; Oncogene 2000, 19:3003,J. Cell Biol.1997,138:913.). Using Western blotting techniques, the ability of205P1B5 to regulate these pathways is examined. Cells expressing 205P1B5and cells lacking these genes are either left untreated or stimulatedwith ions, channel activators, or antibodies. Cell lysates are analyzedusing anti-phospho-specific antibodies (Cell Signaling, Santa CruzBiotechnology) in order to detect phosphorylation and regulation of ERK,p38, AKT, P13K, PLC and other signaling molecules,

When 205P1B5 plays a role in the regulation of signaling pathways,whether individually or communally, it is used as a target fordiagnostic, preventative and therapeutic purposes.

To confirm that 205P1B5 directly or indirectly activates known signaltransduction pathways in cells, luciferase (luc) based transcriptionalreporter assays are carried out in cells expressing individual genes.These transcriptional reporters contain consensus-binding sites forknown transcription factors that lie downstream of well-characterizedsignal transduction pathways. The reporters and examples of theseassociated transcription factors, signal transduction pathways, andactivation stimuli are listed below.

-   1. NFkB-luc, NFkB/Rel; Ik-kinase/SAPK; growth/apoptosis/stress-   2. SRE-luc, SRF/TCF/ELK1; MAPK/SAPK; growth/differentiation-   3. AP-1-luc, FOS/JUN; MAPK/SAPK/PKC; growth/apoptosis/stress-   4. ARE-luc, androgen receptor; steroids/MAPK;    growth/differentiation/apoptosis-   5. p53-luc, p53; SAPK; growth/differentiation/apoptosis-   6. CRE-luc, CREB/ATF2; PKA/p38; growth/apoptosis/stress

Gene-mediated effects are assayed in cells showing mRNA expression.Luciferase reporter plasmids can be introduced by lipid-mediatedtransfection (TFX-50, Promega). Luciferase activity, an indicator ofrelative transcriptional activity, is measured by incubation of cellextracts with luciferin substrate and luminescence of the reaction ismonitored in a luminometer. Signaling pathways activated by 205P1B5 aremapped and used for the identification and validation of therapeutictargets. When these genes are involved in cell signaling, they are usedas targets for diagnostic, preventative and therapeutic purposes.

Example 39 Involvement in Tumor Progression

205P1B5 can contribute to the growth of cancer cells. The role of205P1B5 in tumor growth is investigated in a variety of primary andtransfected cell lines including prostate, colon, bladder and kidneycell lines as well as NIH 3T3 cells engineered to stably express205P1B5. Parental cells lacking our 205P1B5 and cells expressing thegene are evaluated for cell growth using a well-documented proliferationassay (Fraser S P, Grimes J. A, Djamgoz M B. Prostate. 2000;44:61,Johnson D E, Ochieng J, Evans S L. Anticancer Drugs. 1996, 7:288). Theproliferation of control 3T3 and 3T3-205P1B5 were compared using anAlamar blue assay. Control and 205P1B5 expressing cells were grown in0.5% or 10% FBS and analyzed after 48 and 72 hours. As shown in FIG. 16,expression of 205P1B5 enhanced the proliferation of NIH 3T3 cells stablyexpressing 205P1B5. These results indicate that 205P1B5 plays a criticalrole in tumor cell growth

To confirm the role of 205P1B5 in the transformation process, the effectof 205P1B5 in colony forming assays is evaluated. Parental NIH3T3 cellslacking 205P1B5 are compared to NIH-3T3 cells expressing 205P1B5, usinga soft agar assay under stringent and more permissive conditions (SongZ. et al. Cancer Res. 2000; 60:6730). It is found that 205P1B5 causescellular transformation.

To confirm the role of 205P1B5 in invasion and metastasis of cancercells, a well-established Transwell Insert System assay (BectonDickinson) (Cancer Res. 1999; 59:6010) is used. Control cells, includingprostate, colon, bladder and kidney cell lines lacking 205P1B5 arecompared to cells expressing 205P1B5. Cells are loaded with thefluorescent dye, calcein, and plated in the top well of the Transwellinsert coated with a basement membrane analog. Invasion is determined byfluorescence of cells in the lower chamber relative to the fluorescenceof the entire cell population. It is found that 205P1B5 causes invasion.

205P1B5 can also play a role in cell cycle and apoptosis. Parental cellsand cells expressing 205P1B5 are compared for differences in cell cycleregulation using a well-established BrdU assay (Abdel-Malek Z A. J CellPhysiol. 1988, 136:247). In short, cells are grown under both optimal(full serum) and limiting (low serum) conditions are labeled with BrdUand stained with anti-BrdU Ab and propidiumn iodide. Cells are analyzedfor entry into the G1, S, and G2M phases of the cell cycle.Alternatively, the effect of stress on apoptosis is evaluated in controlparental cells and cells expressing genes under consideration, includingnormal and tumor prostate, colon and lung cells. Engineered and parentalcells are treated with various chemotherapeutic agents, such asetoposide, flutamide, etc., and protein synthesis inhibitors, such ascycloheximide. Cells are stained with annexin V-FITC and cell death ismeasured by FACS analysis. It is found that 205P1B5 adversely affectscell cycling and apoptosis.

The function of 205P1B5 is evaluated using anti-sense RNA technologycoupled to the various functional assays described above, e.g. growth,invasion and migration. Anti-sense RNA oligonucleotides can beintroduced into 205P1B5 expressing cells, thereby preventing theexpression of 205P1B5. Control and anti-sense containing cells areanalyzed for proliferation, invasion, migration, apoptotic andtranscriptional potential. The local as well as systemic effects of theloss of 205P1B5 expression are evaluated. It is found that 205P1B5expression adversely impacts properties such as proliferation, invasion,migration, apoptosis and transcription.

When 205P1B5 plays a role in cell growth, transformation, invasion orapoptosis, it is used as a target for diagnostic, preventative andtherapeutic purposes.

Example 40 Regulation of Transcription

Several ion transporters have been shown to play a role intranscriptional regulation of eukaryotic genes. Regulation of geneexpression can be evaluated by studying gene expression in cellsexpressing or lacking 205P1B5. For this purpose, two types ofexperiments are performed. In the first set of experiments, RNA fromparental and gene-expressing cells are extracted and hybridized tocommercially available gene arrays (Clontech) (Smid-Koopman, E, et al.Br. J. Cancer 2000 83:246). Resting cells as well as cells treated withions, FBS or androgen are compared. Differentially expressed genes areidentified in accordance with procedures known in the art. Thedifferentially expressed genes are then mapped to biological pathways(see, e.g., Chen K et al. Thyroid. 2001. 11:41.).

In the second set of experiments, specific transcriptional pathwayactivation is evaluated using commercially available (Stratagene)luciferase reporter constructs including: NFkB-luc, SRE-luc, ELK1-luc,ARE-luc, p53-luc, and CRE-luc. These transcriptional reporters containconsensus binding sites for known transcription factors that liedownstream of well-characterized signal transduction pathways, andrepresent a good tool to ascertain pathway activation and screen forpositive and negative modulators of pathway activation.

Thus, it is found that 205P1B5 adversely impacts gene regulation, and itis used as a target for diagnostic, prognostic, preventative andtherapeutic purposes.

Example 41 Subcellular Localization and Cell Binding

Based on bioinformatic analysis and function, 205P1B5 is indicated to belocated at the cell surface. The cellular location of 205P1B5 isconfirmed using subcellular fractionation techniques widely used incellular biology (Storrie B, et al. Methods Enzymol. 1990;182:203-25). Avariety of cell lines, including prostate, kidney and bladder cell linescan be separated into nuclear, cytosolic and membrane fractions. Geneexpression and location in nuclei, heavy membranes (lysosomes,peroxisomes, and mitochondria), light membranes (plasma membrane andendoplasmic reticulum), and soluble protein fractions are tested usingWestern blotting techniques.

Alternatively, 293T cells are transfected with an expression vectorencoding 205P1B5 HIS-tagged (PCDNA 3.1 MYC/HIS, Invitrogen), and thesubcellular localization of 205P1B5 is confirmed by immunofluorescence.Alternatively, the location of the HIS-tagged 205P1B5 is followed byWestern blotting to confirm the cell surface localization

When 205P1B5 is localized to specific subcellular locale, such as thecell surface, it is used as a target for diagnostic, preventative andtherapeutic purposes as appreciated by one of ordinary skill in the art.

Example 42 Protein and Ion Transporter Function

Based on bioinformatic analysis, 205P1B5 is indicated to function as atransporter. To confirm that 205P1B5 functions as an ion channel, FACSanalysis and electrophysiology techniques are used (Gergely L, Cook L,Agnello V. Clin Diagn Lab Immunol. 1997;4:70; Skryma R. et al. J.Physiol. 2000, 527: 71). Using FACS analysis and commercially availableindicators (Molecular Probes), parental cells and cells expressing205P1B5 are compared for their ability to transport calcium, sodium andpotassium. Prostate, colon, bladder and kidney normal and tumor celllines are used in these studies. For example cells loaded with calciumresponsive indicators such as Fluo4 and Fura red are incubated in thepresence or absence of ions and analyzed by flow cytometry.

Information derived from these procedures confirms important mechanismsby which cancer cells are regulated by 205P1B5. 205P1B5 regulatesprostate cancer growth by regulating intracellular levels of calcium Ofnote, calcium channel inhibitors have been reported to induce the deathof certain cancer cells, including prostate cancer cell lines (Batra S,Popper L D, Hartley-Asp B. Prostate. 1991,19: 299).

Using a modified rhodamine retention assay (Davies J. et al. Science2000, 290:2295; Leith C et al. Blood 1995, 86:2329) it is confirmed that205P1B5 functions as a protein transporter. Cell lines, such asprostate, colon, bladder and kidney cancer and normal cells, expressingor lacking 205P1B5 are loaded with Calcein AM (Molecular Probes). Cellsare examined over time for dye transport using a fluorescent microscopeor fluorometer. Quantitation is performed using a fluorometer (Hollo Z.et al., Biochim Biophys. Acta. 1994. 1191:384). Information obtainedfrom such experiments determines that 205P1B5 serves to extrudechemotherapeutic drugs, such as doxorubicin, paclitaxel, etoposide,etc., from tumor cells, thereby lowering drug content and reducing tumorresponsiveness to treatment. Such a system also determines that 205P1B5functions in transporting small molecules.

When 205P1B5 functions as a transporter, it is used as a target forpreventative, prognostic, diagnostic and/or therapeutic purposes as wellas drug sensitivity/resistance.

Using electrophysiology, uninjected oocytes and oocytes injected withgene-specific cRNA are compared for ion channel activity. Patch/voltageclamp assays are performed on oocytes in the presence or absence ofselected ions, including calcium, potassium sodium, etc. Ion channelactivators (such as cAMP/GMP, forskolin, TPA, etc.) and inhibitors (suchas calcicludine, conotoxin, TEA, tetrodotoxin, etc.) confirm that205P1B5 functions as an ion channel (Schweitz H. et al. Proc. Natl.Acad. Sci. 1994. 91:878; Skryma R et al. Prostate. 1997. 33:112). Usingsimilar techniques, it was recently demonstrated that hCaT inducescalcium flux in 293T cells (Wissenbach, U., et al. J. Biol. Chem. 2001,276: 19461). The magnitude of the flux shown in this paper was similarto the one observed in figure A, where hCaT was expressed in prostatecancer cells.

Thus, 205P1B5 functions as an ion channel, and it is used as a targetfor diagnostic, preventative, prognostic and therapeutic purposes.

Example 43 Involvement in Cell-Cell Communication

Cell-cell communication is essential in maintaining organ integrity andhomeostasis, both of which become dysregulated during tumor formationand progression. Intercellular communications can be measured using twotypes of assays (J. Biol. Chem. 2000, 275:25207). In the first assay,cells loaded with a fluorescent dye are incubated in the presence ofunlabeled recipient cells and the cell populations are examined underfluorescent microscopy. This qualitative assay measures the exchange ofdye between adjacent cells. In the second assay system, donor andrecipient cell populations are treated as above and quantitativemeasurements of the recipient cell population are performed by FACSanalysis. Using these two assay systems, cells expressing or lacking205P1B5 are compared and it is determined that 205P1B5 adversely impactscellular communications. This assay also identifies small moleculesand/or specific antibodies that modulate cell-cell communication.

Thus, 205P1B5 adversely impacts cell-cell communication, and it is usedas a target for diagnostic, prognostics, preventative and therapeuticpurposes.

Example 44 Protein-Protein Interaction

Several ion transporters have been shown to interact with otherproteins, thereby forming a protein complex that can regulate iontransport, cell division, gene transcription, and cell transformation(Biochem Biophys Res Commun. 2000, 277: 61 1; J Biol Chem. 1999; 274:20812). Using immunoprecipitation techniques as well as two yeast hybridsystems, proteins that associate with 205P1B5 are identified.Immunoprecipitates from cells expressing 205P1B5 and cells lacking205P1B5 are compared for specific protein-protein associations. 205P1B5may also associate with, for example, effector molecules, such asadaptor proteins, SNARE proteins, signaling molecules, syntaxins, ATPasesubunits, etc. (3 Biol Chem. 1999; 274: 20812; Proc Natl Acad Sci USA1998, 95:14523). Studies comparing 205P1B5 positive and 205P1B5 negativecells as well as studies comparing unstimulated/resting cells and cellstreated with epithelial cell activators, such as cytokines, growthfactors, androgen and anti-integrin Ab reveal unique interactions.

In addition, protein-protein interactions are confirmed using two yeasthybrid methodologies (see, e.g., Curr Opin Chem Biol. 1999, 3:64). Avector carrying a library of proteins fused to the activation domain ofa transcription factor is introduced into yeast expressing a205P1B5-DNA-binding domain fusion protein and a reporter construct.Protein-protein interaction is detected by colorimetric reporteractivity. Specific association with effector molecules and transcriptionfactors directs one of skill to the mode of action of 205P1B5, and thusidentifies therapeutic, preventative and/or diagnostic targets forcancer. This and similar assays are also used to identify and screen forsmall molecules that interact with 205P1B5.

Thus, 205P1B5 associates with proteins or small molecules and is used asa target for diagnostic, prognostic, preventative and therapeuticpurposes.

Example 45 Transcript Variants of 20SP1B5

Transcript variants are variants of matured mRNA from the same gene byalternative transcription or alternative splicing. Alternativetranscripts are transcripts from the same gene but start transcriptionat different points. Splice variants are mRNA variants spliceddifferently from the same transcript In eukaryotes, when a multi-exongene is transcribed from genomic DNA, the initial RNA is spliced toproduce functional mRNA, which has only exons and is used fortranslation into an amino acid sequence. Accordingly, a given gene canhave zero to many alternative transcripts and each transcript can havezero to many splice variants. Each transcript variant has a unique exonmakeup, and can have different coding and/or non-coding (5′ or 3′ end)portions, from the original transcript. Transcript variants can code forsimilar or different proteins with the same or a similar function or mayencode proteins with different functions, and may be expressed in thesame tissue at the same time, or at different tissue, or at differenttimes, proteins encoded by transcript variants can have similar ordifferent cellular or extracellular localizations, i.e., be secreted.

Transcript variants are identified by a variety of art-accepted methods.For example, alternative transcripts and splice variants are identifiedin a full-length cloning experiment, or by use of full-length transcriptand EST sequences. First, all human ESTs were grouped into clusterswhich show direct or indirect identity with each other. Second, ESTs inthe same cluster were further grouped into sub-clusters and assembledinto a consensus sequence. The original gene sequence is compared to theconsensus sequence(s) or other full-length sequences. Each consensussequence is a potential splice variant for that gene (see, e.g., Kan,Z., et al., Gene structure prediction and alternative splicing analysisusing genomically aligned ESTs, Genome Research, 2001 May,11(5):889-900). Even when a variant is identified that is not afull-length clone, that portion of the variant is useful for antigengeneration and for further cloning of the full-length splice variant,using techniques known in the art.

Moreover, computer programs are available in the art that identifytranscript variants based on genomic sequences. Genomic-based transcriptvariant identification programs include FgenesH (A. Salamov and V.Solovyev, “Ab initio gene finding in Drosophila genomic DNA,” GenomeResearch. 2000 Apr. 10(4):516-22); Grail (world wide web URL//compbio.ornl.gov/Grail-bin/EmptyGrailForm) and GenScan (world wide webURL//genes.mit.edu/GENSCAN.html). For a general discussion of splicevariant identification protocols see., e.g., Southan, C., A genomicperspective on human proteases, FEBS Lett. 2001 Jun. 8; 498(2-3):214-8;de Souza, S. J., et al., Identification of human chromosome 22transcribed sequences with ORF expressed sequence tags, Proc. Natl AcadSci USA. 2000 Nov. 7; 97(23):12690-3.

To further confirm the parameters of a transcript variant, a variety oftechniques are available in the art, such as full-length cloning,proteomic validation, PCR-based validation, and 5′ RACE validation, etc.(see e.g., Proteomic Validation: Brennan, S. O., et al., Albumin bankspeninsula: a new termination variant characterized by electrospray massspectrometry, Biochem Biophys Acta. 1999 Aug. 17;1433(1-2):321-6;Ferranti P, et al., Differential splicing of pre-messenger RNA producesmultiple forms of mature caprine alpha(s1)-casein, Eur J Biochem. 1997Oct. 1;249(1):1-7. For PCR-based Validation: Wellmann S, et al.,Specific reverse transcription-PCR quantification of vascularendothelial growth factor (VEGF) splice variants by LightCyclertechnology, Clin Chem. 2001 April; 47(4):654-60; Jia, H. P., et al.,Discovery of new human beta-defensins using a genomics-based approach,Gene. 2001 Jan. 24; 263(1-2):211-8. For PCR-based and 5′ RACEValidation: Brigle, K. E., et al., Organization of the murine reducedfolate carrier gene and identification of variant splice forms, BiochemBiophys Acta. 1997 Aug. 7; 1353(2): 191-8).

It is known in the art that genomic regions are modulated in cancers.When the genomic region, to which a gene maps, is modulated in aparticular cancer, the alternative transcripts or splice variants of thegene are modulated as well. Disclosed herein is that 205P1B5 has aparticular expression profile related to cancer. Alternative transcriptsand splice variants of 205P1B5 may also be involved in cancers in thesame or different tissues, thus serving as tumor-associatedmarkers/antigens.

Example 46 Singe Nucleotide Polymorphisms of 205P1B5

A Single Nucleotide Polymorphism (SNP) is a single base pair variationin nucleotide sequences. At a specific point of the genome, there arefour possible nucleotide base pairs: A/T, C/G, G/C and T/A. Genotyperefers to the base pair make-up of one or more spots in the genome of anindividual, while haplotype refers to base pair make-up of more than onevaried spots on the same DNA molecule (chromosome in higher organism).SNPs that occur on a cDNA are called cSNPs. These cSNPs may change aminoacids of the protein encoded by the gene and thus change the functionsof the protein. Some SNPs cause inherited diseases and some others,contribute to quantitative variations in phenotype and reactions toenvironmental factors including diet and drugs among individuals.Therefore, SNPs and/or combinations of alleles (called haplotypes) havemany applications including diagnosis of inherited diseases,determination of drug reactions and dosage, identification of genesresponsible for diseases and discovery of genetic relationship betweenindividuals (P. Nowotny, 3. M. Kwon and A. M. Goate, “SNP analysis todissect human traits,” Curr. Opin. Neurobiol. 2001 Oct.; 11(5):637-641;M. Pirmohamed and B. K. Park, “Genetic susceptibility to adverse drugreactions,” Trends Pharmacol. Sci. 2001 June; 22(6):298-305; J. H.Riley, C. J. Allan, E. Lai and A. Roses, “The use of single nucleotidepolymorphisms in the isolation of common disease genes,”Pharmacogenomics. 2000 February; 1(1):39-47; R. Judson, J. C. Stephensand A. Windemuth, “The predictive power of haplotypes in clinicalresponse,” Pharmacogenomics. 2000 February; 1(1):15-26).

SNPs are identified by a variety of art-accepted methods (P. Bean, “Thepromising voyage of SNP target discovery,” Am. Clin. Lab. 2001October-November; 20(9):18-20; K. M. Weiss, “In search of humanvariation,” Genome Res. 1998 July; 8(7):691-697; M. M. She, “Enablinglarge-scale pharmacogenetic studies by high-throughput mutationdetection and genotyping technologies,” Clin. Chem. 2001 February;47(2):164-172). For example, SNPs are identified by sequencing DNAfragments that show polymorphism by gel-based methods such asrestriction fragment length polymorphism (RFLP) and denaturing gradientgel electrophoresis (DGGE). They can also be discovered by directsequencing of DNA samples pooled from different individuals or bycomparing sequences from different DNA samples. With the rapidaccumulation of sequence data in public and private databases, one candiscover SNPs by comparing sequences using computer programs (Z. Gu, L.Hillier and P. Y. Kwok, “Single nucleotide polymorphism hunting incyberspace,” Hum. Mutat. 1998; 12(4):221-225). SNPs can be verified andgenotype or haplotype of an individual can be determined by a variety ofmethods including direct sequencing and high throughput microarrays (P.Y. Kwok, “Methods for genotyping single nucleotide polymorphisms,” Annu.Rev. Genomics Hum. Genet. 2001; 2:235-258; M. Kokoris, K. Dix, K.Moynihan, J. Mathis, B. Erwin, P. Grass, B. Hines and A. Duesterhoeft,“High-throughput SNP genotyping with the Masscode system,” Mol. Diagn.2000 December; 5(4):329-340).

Two variants are identified for 205P1B5. These are set forth in FIG. 2and FIG. 3.

Throughout this application, various website data content, publications,patent applications and patents are referenced. (Websites are referencedby their Uniform Resource Locator, or URL, addresses on the World WideWeb.) The disclosures of each of these references are herebyincorporated by reference herein in their entireties.

The present invention is not to be limited in scope by the embodimentsdisclosed herein, which are intended as single illustrations ofindividual aspects of the invention, and any that are functionallyequivalent are within the scope of the invention. Various modificationsto the models and methods of the invention, in addition to thosedescribed herein, will become apparent to those skilled in the art fromthe foregoing description and teachings, and are similarly intended tofall within the scope of the invention. Such modifications or otherembodiments can be practiced without departing from the true scope andspirit of the invention.

TABLES

TABLE I: Tissues that Express 205P1B5 When Malignant Prostate

TABLE II AMINO ACID ABBREVIATIONS SINGLE LETTER THREE LETTER FULL NAME FPhe phenylalanine L Leu leucine S Ser serine Y Tyr tyrosine C Cyscysteine W Trp tryptophan P Pro proline H His histidine Q Gln glutamineR Arg arginine I Ile isoleucine M Met methionine T Thr threonine N Asnasparagine K Lys lysine V Val valine A Ala alanine D Asp aspartic acid EGlu glutamic acid G Gly glycine

TABLE III AMINO ACID SUBSTITUTION MATRIX Adapted from the GCG Software9.0 BLOSUM62 amino acid substitution matrix (block substitution matrix).The higher the value, the more likely a substitution is found inrelated, natural proteins. (See world wide web URLikp.unibe.ch/manual/blosum62.html) A C D E F G H I K L M N P Q R S T V WY . 4 0 −2 −1 −2 0 −2 −1 −1 −1 −1 −2 −1 −1 −1 1 0 0 −3 −2 A 9 −3 −4 −2−3 −3 −1 −3 −1 −1 −3 −3 −3 −3 −1 −1 −1 −2 −2 C 6 2 −3 −1 −1 −3 −1 −4 −31 −1 0 −2 0 −1 −3 −4 −3 D 5 −3 −2 0 −3 1 −3 −2 0 −1 2 0 0 −1 −2 −3 −2 E6 −3 −1 0 −3 0 0 −3 −4 −3 −3 −2 −2 −1 1 3 F 6 −2 −4 −2 −4 −3 0 −2 −2 −20 −2 −3 −2 −3 G 8 −3 −1 −3 −2 1 −2 0 0 −1 −2 −3 −2 2 H 4 −3 2 1 −3 −3 −3−3 −2 −1 3 −3 −1 I 5 −2 −1 0 −1 1 2 0 −1 −2 −3 −2 K 4 2 −3 −3 −2 −2 −2−1 1 −2 −1 L 5 −2 −2 0 −1 −1 −1 1 −1 −1 M 6 −2 0 0 1 0 −3 −4 −2 N 7 −1−2 −1 −1 −2 −4 −3 P 5 1 0 −1 −2 −2 −1 Q 5 −1 −1 −3 −3 −2 R 4 1 −2 −3 −2S 5 0 −2 −2 T 4 −3 −1 V 11 2 W 7 Y

TABLE IV A POSITION POSITION POSITION C Terminus 2 (Primary 3 (Primary(Primary Anchor) Anchor) Anchor) SUPERMOTIFS A1 TI LVMS FWY A2 LIVM ATQIV MATL A3 VSMA TLI RK A24 YF WIVLMT FI YWLM B7 P VILF MWYA B27 RHK FYLWMIVA B44 E D FWYLIMVA B58 ATS FWY LIVMA B62 QL IVMP FWYMIVLA MOTIFS A1TSM Y A1 DE AS Y A2.1 LM VQIAT V LIMAT A3 LMVISATF CGD KYR HFA A11VTMLISAGN CDF K RYH A24 YF WM FLIW A*3101 MVT ALIS R K A*3301 MVALF ISTRK A*6801 AVT MSLI RK B*0702 P LMF WYAIV B*3501 P LMFWY IVA B51 P LIVFWYAM B*5301 P IMFWY ALV B*5401 P ATIV LMFWY Bolded residues arepreferred, italicized residues are less preferred: A peptide isconsidered motif-bearing if it has primary anchors at each primaryanchor position for a motif or supermotif as specified in the abovetable.

TABLE IV (B) HLA CLASS II SUPERMOTIF 1 6 9 W, F, Y, V, I, L A, V, I, L,P, C, S, T A, V, I, L, C, S, T, M, Y

TABLE IV (C) HLA Class II Motifs MOTIFS 1° anchor 1 2 3 4 5 1° anchor 67 8 9 DR4 preferred FMYLIVW M T I VSTCPALIM MH MH deleterious W R WDEDR1 preferred MFLIVWY PAMQ VMATSPLIC M AVM deleterious C CH FD CWD GDE DDR7 preferred MFLIVWY M W A IVMSACTPL M IV deleterious C G GRD N G DR3MOTIFS 1° anchor 1 2 3 1° anchor 4 5 1° anchor 6 motif a LIVMFY Dpreferred motif b LIVMFAY DNQEST KRH preferred DR MFLIVWY VMSTACPLISupermotif Italicized residues indicate less preferred or “tolerated”residues.

TABLE IV (D) HLA Class I Supermotifs POSITION SUPERMOTIFS 1 2 3 4 5 6 78 C-terminus A1 1° Anchor 1° Anchor TILVMS FWY A2 1° Anchor 1° AnchorLIVMATQ LIVMAT A3 preferred 1° Anchor YFW (4/5) YFW (3/5) YFW (4/5) P(4/5) 1° Anchor VSMATLI RK delete- DE (3/5); DE (4/5) rious P (5/5) A241° Anchor 1° Anchor YFWIVLMT FIYWLM B7 preferred FWY (5/5) 1° Anchor FWY(4/5) FWY (3/5) 1° Anchor LIVM (3/5) P VILFMWYA delete- DE (3/5); DE(3/5) G (4/5) QN (4/5) DE (4/5) rious P (5/5); G (4/5); A (3/5); QN(3/5) B27 1° Anchor 1° Anchor RHK FYLWMIVA B44 1° Anchor 1° Anchor EDFWYLIMVA B58 1° Anchor 1° Anchor ATS FWYLIVMA B62 1° Anchor 1° AnchorQLIVMP FWYMIVLA Italicized residues indicate less preferred or“tolerated” residues

TABLE IV (E) HLA Class I Motifs POSITION: 9 or 1 2 3 4 5 6 7 8C-terminus C-terminus A1 preferred GFY 1° Anchor DEA YFW P DEQN YFW1° Anchor 9-mer W STM Y deleterious DE RHKLIVMP A G A A1 preferred GRHKASTCLIVM 1° Anchor GSTC ASTC LIVM DE 1° Anchor 9-mer DEAS Y deleteriousA RHKDEPYFW DE PQN RHK PG GP A1 preferred YFW 1° Anchor DEAQN A YFWQNPASTC GDE P 1° Anchor 10-mer STM Y deleterious GP RHKGLIVM DE RHK QNARHKYFW RHK A A1 preferred YFW STCLIVM 1° Anchor A YFW PG G YFW 1° Anchor10-mer DEAS Y deleterious RHK RHKDEPYFW P G PRHK QN A2.1 preferred YFW1° Anchor YFW STC YFW A P 1° Anchor 9-mer LMIVQAT VLIMAT deleterious DEPDERKH RKH DERKH Italicized residues indicate less preferred or“tolerated” residues

TABLE IV (E) HLA Class I Motifs, POSITION: 1 2 3 4 5 6 7 8 9 C-terminusA2.1 preferred AYFW 1° Anchor LVIM G G FYWLVIM 1° Anchor 10-mer LMIVQATVLIMAT deleterious DEP DE RKHA P RKH DERKH RKH A3 preferred RHK 1°Anchor YFW PRHKYFW A YFW P 1° Anchor LMVISATFCGD KYRHFA deleterious DEPDE A11 preferred A 1° Anchor YFW YFW A YFW YFW P 1° Anchor VTLMISAGNCDFKRYH deleterious DEP A G A24 preferred YFWRHK 1° Anchor STC YFW YFW1° Anchor 9-mer YFWM FLIW deleterious DEG DE G QNP DERHK G AQN A24preferred 1° Anchor P YFWP P 1° Anchor 10-mer YFWM FLIW deleterious GDEQN RHK DE A QN DEA A3101 preferred RHK 1° Anchor YFW P YFW YFW AP1° Anchor MVTALIS RK deleterious DEP DE ADE DE DE DE A3301 preferred 1°Anchor YFW AYFW 1° Anchor MVALFIST RK deleterious GP DE Italicizedresidues indicate less preferred or “tolerated” residues

TABLE IV (E) HLA Class I Motifs, POSITION: C- 1 2 3 4 5 6 7 8 9 TerminusA6801 preferred YFWSTC 1° Anchor YFWLIVM YFW P 1° Anchor AVTMSLI RKdeleterious GP DEG RHK A B0702 preferred RHKFWY 1° Anchor RHK RHK RHKRHK PA 1° Anchor P LMFWYAIV deleterious DEQNP DEP DE DE GDE QN DE B3501preferred FWYLIVM 1° Anchor FWY FWY 1° Anchor P LMFWYIVA deleterious AGPG G B51 preferred LIVMFWY 1° Anchor FWY STC FWY G FWY 1° Anchor PLIVFWYAM deleterious AGPDERHKSTC DE G DEQN GDE B5301 preferred LIVMFWY1° Anchor FWY STC FWY LIVMFWY FWY 1° Anchor P IMFWYALV deleterious AGPQNG RHKQN DE B5401 preferred FWY 1° Anchor FWYLIVM LIVM ALIVM FWYAP 1°Anchor P ATIVLMFWY deleterious GPQNDE GDESTC RHKDE DE QNDGE DEItalicized residues indicate less preferred or “tolerated” residues. Theinformation in this Table is specific for 9-mers unless otherwisespecified.

TABLE V HLA Peptide Scoring Results - 205P1B5 - A1, 9-mers Score(Estimate of Half Time of Subsequence Disassociation of a Start ResidueMolecule Containing Rank Position Listing This Subsequence) Seq. ID# 1184 SIDVTFFPF 25.000 1. 2 55 HTETEDRLF 22.500 2. 3 80 TSDVVIVRF 15.0003. 4 57 ETEDRLFKH 11.250 4. 5 215 QMEQTVDLK 9.000 5. 6 323 LVIPLIGEY5.000 6. 7 210 KIDLEQMEQ 2.500 7. 8 314 ITEIIPSTS 2.250 8. 9 386PVELCHPLR 1.800 9. 10 469 ALEGVHYIA 1.800 10. 11 409 DAEEREVVV 1.800 11.12 481 RSEDADSSV 1.350 12. 13 244 KYDCCAEIY 1.250 13. 14 79 NTSDVVIVR1.250 14. 15 255 VTYAFVIRR 1.250 15. 16 328 IGEYLLFTM 1.125 16. 17 280SCLTVLVFY 1.000 17. 18 150 NADGEFAVT 1.000 18. 19 460 LLLSPHMQK 1.00019. 20 248 CAEIYPDVT 0.900 20. 21 416 VVEEEDRWA 0.900 21. 22 450KAEALLQEG 0.900 22. 23 212 DLEQMEQTV 0.900 23. 24 3 PSCPVFLSF 0.750 24.25 115 WSDYKLRWN 0.750 25. 26 279 ISCLTVLVF 0.750 26. 27 455 LQEGELLLS0.675 27. 28 124 PTDFGNITS 0.625 28. 29 232 AIVNATGTY 0.500 29. 30 95LIDVDEKNQ 0.500 30. 31 389 LCHPLRLKL 0.500 31. 32 407 NVDAEEREV 0.50032. 33 372 GCVPRWLLM 0.500 33. 34 97 DVDEKNQMM 0.500 34. 35 360HTMPHWVRG 0.500 35. 36 482 SEDADSSVK 0.500 36. 37 236 ATGTYNSKK 0.50037. 38 172 HWVPPAIYK 0.500 38. 39 272 LIIPCLLIS 0.500 39. 40 140IWIPDIVLY 0.500 40. 41 228 SGEWAIVNA 0.450 41. 42 486 DSSVKEDWK 0.30042. 43 181 SSCSIDVTF 0.300 43. 44 112 KQEWSDYKL 0.270 44. 45 142IPDIVLYNN 0.250 45. 46 499 VIDRIFLWL 0.250 46. 47 5 CPVFLSFTK 0.250 47.48 252 YPDVTYAFV 0.250 48. 49 457 EGELLLSPH 0.225 49. 50 152 DGEFAVTHM0.225 50.

TABLE VI HLA Peptide Scoring Results - 205P1B5 - A1, 10-mers Score(Estimate of Half Time of Subsequence Disassociation of a Start ResidueMolecule Containing Rank Position Listing This Subsequence) Seq. ID# 155 HTETEDRLFK 225.000 51. 2 248 CAEIYPDVTY 90.000 52. 3 481 RSEDADSSVK27.000 53. 4 97 DVDEKNQMMT 2.500 54. 5 57 ETEDRLFKHL 2.250 55. 6 314ITEIIPSTSL 2.250 56. 7 250 EIYPDVTYAF 2.000 57. 8 279 ISCLTVLVFY 1.50058. 9 112 KQEWSDYKLR 1.350 59. 10 236 ATGTYNSKKY 1.250 60. 11 150NADGEFAVTH 1.000 61. 12 170 TVHWVPPAIY 1.000 62. 13 4 SCPVFLSFTK 1.00063. 14 459 ELLLSPHMQK 1.000 64. 15 416 VVEEEDRWAC 0.900 65. 16 26GEEAKRPPPR 0.900 66. 17 486 DSSVKEDWKY 0.750 67. 18 183 CSIDVTFFPF 0.75068. 19 80 TSDVVIVRFG 0.750 69. 20 252 YPDVTYAFVI 0.625 70. 21 95LIDVDEKNQM 0.500 71. 22 476 IADHLRSEDA 0.500 72. 23 278 LISCLTVLVF 0.50073. 24 484 DADSSVKEDW 0.500 74. 25 372 GCVPRWLLMN 0.500 75. 26 322SLVIPLIGEY 0.500 76. 27 499 VIDRIFLWLF 0.500 77. 28 515 GTIGLFLPPF 0.50078. 29 254 DVTYAFVIRR 0.500 79. 30 407 NVDAEEREVV 0.500 80. 31 316EIIPSTSLVI 0.500 81. 32 210 KIDLEQMEQT 0.500 82. 33 79 NTSDVVIVRF 0.50083. 34 219 TVDLKDYWES 0.500 84. 35 272 LIIPCLLISC 0.500 85. 36 231WAIVNATGTY 0.500 86. 37 21 LTPAGGEEAK 0.500 87. 38 386 PVELCHPLRL 0.45088. 39 457 EGELLLSPHM 0.450 89. 40 469 ALEGVHYIAD 0.450 90. 41 290PSDCGEKITL 0.375 91. 42 446 ASGPKAEALL 0.300 92. 43 180 KSSCSIDVTF 0.30093. 44 136 PSEMIWIPDI 0.270 94. 45 360 HTMPHWVRGA 0.250 95. 46 358STHTMPHWVR 0.250 96. 47 157 VTHMTKAHLF 0.250 97. 48 339 VTLSIVITVF 0.25098. 49 259 FVIRRLPLFY 0.250 99. 50 228 SGEWAIVNAT 0.225 100.

TABLE VII HLA Peptide Scoring Results - 205P1B5 - A2, 9-mers Score(Estimate of Half Time of Subsequence Disassociation of a Start ResidueMolecule Containing Rank Position Listing This Subsequence) Seq. ID# 1331 YLLFTMIFV 14974.754 101. 2 506 WLFIIVCFL 4599.389 102. 3 281CLTVLVFYL 1567.359 103. 4 303 VLLSLTVFL 739.032 104. 5 504 FLWLFIIVC679.693 105. 6 396 KLSPSYHWL 616.839 106. 7 513 FLGTIGLFL 540.469 107. 8304 LLSLTVFLL 484.457 108. 9 103 QMMTTNVWL 313.968 109. 10 276 CLLISCLTV257.342 110. 11 332 LLFTMIFVT 256.368 111. 12 520 FLPPFLAGM 199.733 112.13 454 LLQEGELLL 148.896 113. 14 344 VITVFVLNV 142.093 114. 15 13KLSLWWLLL 127.106 115. 16 518 GLFLPPFLA 106.613 116. 17 8 FLSFTKLSL98.267 117. 18 468 KALEGVHYI 97.322 118. 19 306 SLTVFLLLI 91.183 119. 20327 LIGEYLLFT 90.344 120. 21 379 LMNRPPPPV 85.394 121. 22 497 AMVIDRIFL84.856 122. 23 336 MIFVTLSIV 56.725 123. 24 370 LLGCVPRWL 39.948 124. 25453 ALLQEGELL 38.730 125. 26 310 FLLLITEII 35.673 126. 27 277 LLISCLTVL34.246 127. 28 502 RIFLWLFII 29.030 128. 29 166 FSTGTVHWV 26.419 129. 30499 VIDRIFLWL 20.873 130. 31 461 LLSPHMQKA 19.425 131. 32 139 MIWIPDIVL16.993 132. 33 313 LITEIIPST 16.426 133. 34 278 LISCLTVLV 16.258 134. 35342 SIVITVFVL 16.065 135. 36 339 VTLSIVITV 13.975 136. 37 324 VIPLIGEYL13.457 137. 38 134 RVPSEMIWI 11.548 138. 39 149 NNADGEFAV 10.797 139. 40271 NLIIPCLLI 10.433 140. 41 296 KITLCISVL 9.695 141. 42 84 VIVRFGLSI9.267 142. 43 508 FIIVCFLGT 8.955 143. 44 488 SVKEDWKYV 8.165 144. 45335 TMIFVTLSI 7.535 145. 46 48 ALPQGGSHT 7.452 146. 47 252 YPDVTYAFV6.892 147. 48 269 TINLIIPCL 6.756 148. 49 358 STHTMPHWV 5.313 149. 50517 IGLFLPPFL 4.824 150.

TABLE VIII HLA Peptide Scoring Results - 205P1B5 - A2, 10-mers Score(Estimate of Half Time of Subsequence Disassociation of a Start ResidueMolecule Containing Rank Position Listing This Subsequence) Seq. ID# 1303 VLLSLTVFLL 1792.489 151. 2 331 YLLFTMIFVT 693.701 152. 3 378LLMNRPPPPV 437.482 153. 4 340 TLSIVITVFV 382.536 154. 5 370 LLGCVPRWLL272.371 155. 6 332 LLFTMIFVTL 255.302 156. 7 89 GLSIAQLIDV 159.970 157.8 498 MVIDRIFLWL 136.147 158. 9 277 LLISCLTVLV 118.238 159. 10 15SLWWLLLTPA 94.839 160. 11 343 IVITVFVLNV 90.423 161. 12 369 ALLGCVPRWL86.945 162. 13 453 ALLQEGELLL 79.041 163. 14 276 CLLISCLTVL 74.536 164.15 460 LLLSPHMQKA 71.872 165. 16 304 LLSLTVFLLL 69.001 166. 17 338FVTLSIVITV 64.388 167. 18 327 LIGEYLLFTM 63.515 168. 19 13 KLSLWWLLLT59.989 169. 20 298 TLCISVLLSL 49.134 170. 21 335 TMIFVTLSIV 47.369 171.22 102 NQMMTTNVWL 44.076 172. 23 302 SVLLSLTVFL 38.038 173. 24 280SCLTVLVFYL 37.856 174. 25 461 LLSPHMQKAL 36.316 175. 26 312 LLITEIIPST29.137 176. 27 502 RIFLWLFIIV 27.565 177. 28 288 YLPSDCGEKI 23.516 178.29 263 RLPLFYTINL 21.362 179. 30 204 WTYDKAKIDL 17.906 180. 31 516TIGLFLPPFL 16.155 181. 32 148 YNNADGEFAV 12.113 182. 33 273 IIPCLLISCL11.485 183. 34 296 KITLCISVLL 10.281 184. 35 496 VAMVIDRIFL 10.264 185.36 260 VIRRLPLFYT 9.713 186. 37 300 CISVLLSLTV 9.563 187. 38 323LVIPLIGEYL 8.564 188. 39 284 VLVFYLPSDC 8.446 189. 40 308 TVFLLLITEI7.769 190. 41 508 FIIVCFLGTI 7.497 191. 42 506 WLFIIVCFLG 7.356 192. 43306 SLTVFLLLIT 7.027 193. 44 504 FLWLFIIVCF 6.544 194. 45 520 FLPPFLAGMI6.239 195. 46 83 VVIVRFGLSI 5.897 196. 47 257 YAFVIRRLPL 5.050 197. 48324 VIPLIGEYLL 4.993 198. 49 490 KEDWKYVAMV 4.355 199. 50 487 SSVKEDWKYV4.245 200.

TABLE IX HLA Peptide Scoring Results - 205P1B5 - A3, 9-mers Score(Estimate of Half Time of Subsequence Disassociation of a Start ResidueMolecule Containing Rank Position Listing This Subsequence) Seq. ID# 1104 MMTTNVWLK 180.000 201. 2 460 LLLSPHMQK 90.000 202. 3 215 QMEQTVDLK60.000 203. 4 198 KMKFGSWTY 36.000 204. 5 255 VTYAFVIRR 18.000 205. 6506 WLFIIVCFL 13.500 206. 7 518 GLFLPPFLA 13.500 207. 8 13 KLSLWWLLL10.800 208. 9 504 FLWLFIIVC 9.000 209. 10 288 YLPSDCGEK 6.000 210. 11119 KLRWNPTDF 6.000 211. 12 281 CLTVLVFYL 5.400 212. 13 306 SLTVFLLLI5.400 213. 14 304 LLSLTVFLL 5.400 214. 15 340 TLSIVITVF 4.500 215. 16502 RIFLWLFII 4.050 216. 17 110 WLKQEWSDY 4.000 217. 18 236 ATGTYNSKK3.000 218. 19 396 KLSPSYHWL 2.700 219. 20 93 AQLIDVDEK 2.700 220. 21 271NLIIPCLLI 2.700 221. 22 335 TMIFVTLSI 2.700 222. 23 202 GSWTYDKAK 2.250223. 24 332 LLFTMIFVT 2.250 224. 25 79 NTSDVVIVR 1.800 225. 26 184SIDVTFFPF 1.800 226. 27 454 LLQEGELLL 1.800 227. 28 497 AMVIDRIFL 1.800228. 29 513 FLGTIGLFL 1.800 229. 30 310 FLLLITEII 1.350 230. 31 277LLISCLTVL 1.350 231. 32 388 ELCHPLRLK 1.350 232. 33 369 ALLGCVPRW 1.350233. 34 469 ALEGVHYIA 1.350 234. 35 520 FLPPFLAGM 1.350 235. 36 8FLSFTKLSL 1.200 236. 37 164 HLFSTGTVH 1.000 237. 38 190 FPFDQQNCK 1.000238. 39 65 HLFRGYNRW 1.000 239. 40 453 ALLQEGELL 0.900 240. 41 516TIGLFLPPF 0.900 241. 42 132 SLRVPSEMI 0.900 242. 43 5 CPVFLSFTK 0.900243. 44 139 MIWIPDIVL 0.900 244. 45 103 QMMTTNVWL 0.900 245. 46 331YLLFTMIFV 0.900 246. 47 303 VLLSLTVFL 0.900 247. 48 326 PLIGEYLLF 0.900248. 49 342 SIVITVFVL 0.810 249. 50 260 VIRRLPLFY 0.800 250.

TABLE X HLA Peptide Scoring Results - 205P1B5 - A3, 10-mers Score(Estimate of Half Time of Subsequence Disassociation of a Start ResidueMolecule Containing Rank Position Listing This Subsequence) Seq. ID# 1103 QMMTTNVWLK 270.000 251. 2 110 WLKQEWSDYK 60.000 252. 3 459ELLLSPHMQK 27.000 253. 4 504 FLWLFIIVCF 22.500 254. 5 332 LLFTMIFVTL13.500 255. 6 303 VLLSLTVFLL 8.100 256. 7 61 RLFKHLFRGY 6.000 257. 8 441HLHSGASGPK 6.000 258. 9 346 TVFVLNVHHR 6.000 259. 10 304 LLSLTVFLLL5.400 260. 11 263 RLPLFYTINL 3.600 261. 12 515 GTIGLFLPPF 3.038 262. 13146 VLYNNADGEF 3.000 263. 14 139 MIWIPDIVLY 3.000 264. 15 370 LLGCVPRWLL2.700 265. 16 298 TLCISVLLSL 2.700 266. 17 499 VIDRIFLWLF 2.700 267. 1813 KLSLWWLLLT 2.700 268. 19 518 GLFLPPFLAG 2.700 269. 20 322 SLVIPLIGEY2.700 270. 21 254 DVTYAFVIRR 2.160 271. 22 250 EIYPDVTYAF 2.025 272. 2355 HTETEDRLFK 2.000 273. 24 453 ALLQEGELLL 1.800 274. 25 89 GLSIAQLIDV1.800 275. 26 153 GEFAVTHMTK 1.800 276. 27 15 SLWWLLLTPA 1.500 277. 28276 CLLISCLTVL 1.350 278. 29 199 MKFGSWTYDK 1.350 279. 30 497 AMVIDRIFLW1.350 280. 31 214 EQMEQTVDLK 1.215 281. 32 472 GVHYIADHLR 1.200 282. 33259 FVIRRLPLFY 1.200 283. 34 373 CVPRWLLMNR 1.200 284. 35 278 LISCLTVLVF1.200 285. 36 21 LTPAGGEEAK 1.000 286. 37 164 HLFSTGTVHW 1.000 287. 38358 STHTMPHWVR 0.900 288. 39 235 NATGTYNSKK 0.900 289. 40 265 PLFYTINLII0.900 290. 41 394 RLKLSPSYHW 0.900 291. 42 138 EMIWIPDIVL 0.810 292. 43396 KLSPSYHWLE 0.810 293. 44 498 MVIDRIFLWL 0.810 294. 45 331 YLLFTMIFVT0.675 295. 46 336 MIFVTLSIVI 0.600 296. 47 502 RIFLWLFIIV 0.600 297. 48288 YLPSDCGEKI 0.600 298. 49 92 IAQLIDVDEK 0.600 299. 50 170 TVHWVPPAIY0.600 300.

TABLE XI HLA Peptide Scoring Results - 205P1B5 - A11, 9-mers Score(Estimate of Half Time of Subsequence Disassociation of a Start ResidueMolecule Containing Rank Position Listing This Subsequence) Seq. ID# 1460 LLLSPHMQK 1.200 301. 2 200 KFGSWTYDK 1.200 302. 3 236 ATGTYNSKK1.000 303. 4 5 CPVFLSFTK 0.900 304. 5 93 AQLIDVDEK 0.900 305. 6 255VTYAFVIRR 0.800 306. 7 104 MMTTNVWLK 0.800 307. 8 494 KYVAMVIDR 0.720308. 9 190 FPFDQQNCK 0.400 309. 10 288 YLPSDCGEK 0.400 310. 11 215QMEQTVDLK 0.400 311. 12 79 NTSDVVIVR 0.400 312. 13 22 TPAGGEEAK 0.200313. 14 235 NATGTYNSK 0.200 314. 15 368 GALLGCVPR 0.180 315. 16 414EVVVEEEDR 0.180 316. 17 154 EFAVTHMTK 0.120 317. 18 254 DVTYAFVIR 0.120318. 19 56 TETEDRLFK 0.120 319. 20 134 RVPSEMIWI 0.120 320. 21 498MVIDRIFLW 0.090 321. 22 374 VPRWLLMNR 0.080 322. 23 518 GLFLPPFLA 0.072323. 24 502 RIFLWLFII 0.072 324. 25 202 GSWTYDKAK 0.060 325. 26 172HWVPPAIYK 0.060 326. 27 472 GVHYIADHL 0.060 327. 28 482 SEDADSSVK 0.060328. 29 347 VFVLNVHHR 0.060 329. 30 434 GTLCSHGHL 0.045 330. 31 169GTVHWVPPA 0.045 331. 32 346 TVFVLNVHH 0.040 332. 33 386 PVELCHPLR 0.040333. 34 64 KHLFRGYNR 0.036 334. 35 112 KQEWSDYKL 0.036 335. 36 415VVVEEEDRW 0.030 336. 37 323 LVIPLIGEY 0.030 337. 38 339 VTLSIVITV 0.030338. 39 259 FVIRRLPLF 0.030 339. 40 302 SVLLSLTVF 0.030 340. 41 82DVVIVRFGL 0.027 341. 42 198 KMKFGSWTY 0.024 342. 43 13 KLSLWWLLL 0.024343. 44 11 FTKLSLWWL 0.020 344. 45 495 YVAMVIDRI 0.020 345. 46 442LHSGASGPK 0.020 346. 47 170 TVHWVPPAI 0.020 347. 48 111 LKQEWSDYK 0.020348. 49 428 HVAPSVGTL 0.020 349. 50 85 IVRFGLSIA 0.020 350.

TABLE XII HLA Peptide Scoring Results - 205P1B5 - A11, 10-mers Score(Estimate of Half Time of Subsequence Disassociation of a Start ResidueMolecule Containing Rank Position Listing This Subsequence) Seq. ID# 155 HTETEDRLFK 2.000 351. 2 103 QMMTTNVWLK 1.600 352. 3 472 GVHYIADHLR1.200 353. 4 21 LTPAGGEEAK 1.000 354. 5 346 TVFVLNVHHR 0.800 355. 6 373CVPRWLLMNR 0.800 356. 7 153 GEFAVTHMTK 0.720 357. 8 287 FYLPSDCGEK 0.600358. 9 4 SCPVFLSFTK 0.600 359. 10 110 WLKQEWSDYK 0.400 360. 11 358STHTMPHWVR 0.400 361. 12 441 HLHSGASGPK 0.400 362. 13 214 EQMEQTVDLK0.360 363. 14 459 ELLLSPHMQK 0.360 364. 15 254 DVTYAFVIRR 0.240 365. 16189 FFPFDQQNCK 0.200 366. 17 235 NATGTYNSKK 0.200 367. 18 92 IAQLIDVDEK0.200 368. 19 112 KQEWSDYKLR 0.180 369. 20 199 MKFGSWTYDK 0.080 370. 21171 VHWVPPAIYK 0.080 371. 22 66 LFRGYNRWAR 0.080 372. 23 481 RSEDADSSVK0.060 373. 24 343 IVITVFVLNV 0.060 374. 25 259 FVIRRLPLFY 0.060 375. 26498 MVIDRIFLWL 0.060 376. 27 83 VVIVRFGLSI 0.060 377. 28 413 REVVVEEEDR0.054 378. 29 502 RIFLWLFIIV 0.048 379. 30 515 GTIGLFLPPF 0.045 380. 31169 GTVHWVPPAI 0.045 381. 32 434 GTLCSHGHLH 0.045 382. 33 204 WTYDKAKIDL0.040 383. 34 510 IVCFLGTIGL 0.040 384. 35 308 TVFLLLITEI 0.040 385. 36234 VNATGTYNSK 0.040 386. 37 338 FVTLSIVITV 0.040 387. 38 22 TPAGGEEAKR0.040 388. 39 488 SVKEDWKYVA 0.040 389. 40 334 FTMIFVTLSI 0.040 390. 4126 GEEAKRPPPR 0.036 391. 42 323 LVIPLIGEYL 0.030 392. 43 302 SVLLSLTVFL0.030 393. 44 89 GLSIAQLIDV 0.024 394. 45 263 RLPLFYTINL 0.024 395. 46394 RLKLSPSYHW 0.024 396. 47 351 NVHHRSPSTH 0.020 397. 48 11 FTKLSLWWLL0.020 398. 49 365 WVRGALLGCV 0.020 399. 50 407 NVDAEEREVV 0.020 400.

TABLE XIII HLA Peptide Scoring Results - 205P1B5 - A24, 9-mers Score(Estimate of Half Time of Subsequence Disassociation of a Start ResidueMolecule Containing Rank Position Listing This Subsequence) Seq. ID# 1256 TYAFVIRRL 280.000 401. 2 251 IYPDVTYAF 252.000 402. 3 205 TYDKAKIDL200.000 403. 4 147 LYNNADGEF 165.000 404. 5 330 EYLLFTMIF 150.000 405. 687 RFGLSIAQL 40.000 406. 7 333 LFTMIFVTL 33.600 407. 8 258 AFVIRRLPL30.000 408. 9 512 CFLGTIGLF 15.000 409. 10 112 KQEWSDYKL 13.200 410. 11305 LSLTVFLLL 10.080 411. 12 244 KYDCCAEIY 10.000 412. 13 309 VFLLLITEI9.900 413. 14 396 KLSPSYHWL 9.600 414. 15 337 IFVTLSIVI 9.000 415. 16 82DVVIVRFGL 8.400 416. 17 270 INLIIPCLL 8.400 417. 18 324 VIPLIGEYL 8.400418. 19 269 TINLIIPCL 8.400 419. 20 297 ITLCISVLL 8.400 420. 21 299LCISVLLSL 8.400 421. 22 296 KITLCISVL 8.000 422. 23 13 KLSLWWLLL 8.000423. 24 474 HYIADHLRS 7.500 424. 25 69 GYNRWARPV 7.500 425. 26 239TYNSKKYDC 7.500 426. 27 303 VLLSLTVFL 7.200 427. 28 214 EQMEQTVDL 7.200428. 29 517 IGLFLPPFL 7.200 429. 30 454 LLQEGELLL 7.200 430. 31 266LFYTINLII 7.000 431. 32 499 VIDRIFLWL 6.720 432. 33 452 EALLQEGEL 6.600433. 34 389 LCHPLRLKL 6.336 434. 35 434 GTLCSHGHL 6.000 435. 36 342SIVITVFVL 6.000 436. 37 1 MGPSCPVFL 6.000 437. 38 447 SGPKAEALL 6.000438. 39 462 LSPHMQKAL 6.000 439. 40 453 ALLQEGELL 6.000 440. 41 497AMVIDRIFL 6.000 441. 42 264 LPLFYTINL 6.000 442. 43 400 SYHWLESNV 6.000443. 44 117 DYKLRWNPT 6.000 444. 45 325 IPLIGEYLL 6.000 445. 46 224DYWESGEWA 6.000 446. 47 277 LLISCLTVL 6.000 447. 48 103 QMMTTNVWL 6.000448. 49 472 GVHYIADHL 5.600 449. 50 513 FLGTIGLFL 5.600 450.

TABLE XIV HLA Peptide Scoring Results - 205P1B5 - A24, 10-mers Score(Estimate of Half Time of Disassociation of a Start Subsequence MoleculeContaining Rank Position Residue Listing This Subsequence) Seq. ID# 1494 KYVAMVIDRI 210.000 451. 2 224 DYWESGEWAI 60.000 452. 3 512CFLGTIGLFL 42.000 453. 4 7 VFLSFTKLSL 30.000 454. 5 10 SFTKLSLWWL 20.000455. 6 87 RFGLSIAQLI 16.800 456. 7 258 AFVIRRLPLF 15.000 457. 8 498MVIDRIFLWL 12.096 458. 9 263 RLPLFYTINL 12.000 459. 10 296 KITLCISVLL11.200 460. 11 309 VFLLLITEII 10.500 461. 12 57 ETEDRLFKHL 10.368 462.13 323 LVIPLIGEYL 10.080 463. 14 251 IYPDVTYAFV 9.000 464. 15 369ALLGCVPRWL 8.400 465. 16 361 TMPHWVRGAL 8.400 466. 17 268 YTINLIIPCL8.400 467. 18 471 EGVHYIADHL 8.400 468. 19 269 TINLIIPCLL 8.400 469. 20505 LWLFIIVCFL 8.400 470. 21 5 CPVFLSFTKL 7.920 471. 22 239 TYNSKKYDCC7.500 472. 23 147 LYNNADGEFA 7.500 473. 24 330 EYLLFTMIFV 7.500 474. 25273 IIPCLLISCL 7.200 475. 26 302 SVLLSLTVFL 7.200 476. 27 280 SCLTVLVFYL7.200 477. 28 304 LLSLTVFLLL 6.720 478. 29 332 LLFTMIFVTL 6.720 479. 30267 FYTINLIIPC 6.000 480. 31 324 VIPLIGEYLL 6.000 481. 32 453 ALLQEGELLL6.000 482. 33 452 EALLQEGELL 6.000 483. 34 138 EMIWIPDIVL 6.000 484. 35102 NQMMTTNVWL 6.000 485. 36 341 LSIVITVFVL 6.000 486. 37 496 VAMVIDRIFL6.000 487. 38 276 CLLISCLTVL 6.000 488. 39 314 ITEIIPSTSL 6.000 489. 40303 VLLSLTVFLL 6.000 490. 41 255 VTYAFVIRRL 5.600 491. 42 180 KSSCSIDVTF5.600 492. 43 298 TLCISVLLSL 5.600 493. 44 388 ELCHPLRLKL 5.280 494. 45380 MNRPPPPVEL 5.280 495. 46 178 IYKSSCSIDV 5.000 496. 47 446 ASGPKAEALL4.800 497. 48 318 IPSTSLVIPL 4.800 498. 49 11 FTKLSLWWLL 4.800 499. 50516 TIGLFLPPFL 4.800 500.

TABLE XV HLA Peptide Scoring Results - 205P1B5 - B7, 9-mers Score(Estimate of Half Time of Subsequence Disassociation of a Start ResidueMolecule Containing Rank Position Listing This Subsequence) Seq. ID# 1362 MPHWVRGAL 120.000 501. 2 33 PPRAPGDPL 120.000 502. 3 325 IPLIGEYLL80.000 503. 4 274 IPCLLISCL 80.000 504. 5 264 LPLFYTINL 80.000 505. 6 82DVVIVRFGL 30.000 506. 7 472 GVHYIADHL 20.000 507. 8 428 HVAPSVGTL 20.000508. 9 497 AMVIDRIFL 18.000 509. 10 453 ALLQEGELL 12.000 510. 11 103QMMTTNVWL 12.000 511. 12 446 ASGPKAEAL 12.000 512. 13 214 EQMEQTVDL12.000 513. 14 452 EALLQEGEL 12.000 514. 15 371 LGCVPRWLL 9.000 515. 16385 PPVELCHPL 8.000 516. 17 289 LPSDCGEKI 8.000 517. 18 521 LPPFLAGMI8.000 518. 19 77 VPNTSDVVI 8.000 519. 20 139 MIWIPDIVL 6.000 520. 21 389LCHPLRLKL 6.000 521. 22 132 SLRVPSEMI 6.000 522. 23 365 WVRGALLGC 5.000523. 24 85 IVRFGLSIA 5.000 524. 25 396 KLSPSYHWL 4.000 525. 26 297ITLCISVLL 4.000 526. 27 511 VCFLGTIGL 4.000 527. 28 13 KLSLWWLLL 4.000528. 29 281 CLTVLVFYL 4.000 529. 30 277 LLISCLTVL 4.000 530. 31 157VTHMTKAHL 4.000 531. 32 324 VIPLIGEYL 4.000 532. 33 370 LLGCVPRWL 4.000533. 34 11 FTKLSLWWL 4.000 534. 35 296 KITLCISVL 4.000 535. 36 1MGPSCPVFL 4.000 536. 37 299 LCISVLLSL 4.000 537. 38 517 IGLFLPPFL 4.000538. 39 506 WLFIIVCFL 4.000 539. 40 270 INLIIPCLL 4.000 540. 41 8FLSFTKLSL 4.000 541. 42 304 LLSLTVFLL 4.000 542. 43 434 GTLCSHGHL 4.000543. 44 342 SIVITVFVL 4.000 544. 45 305 LSLTVFLLL 4.000 545. 46 447SGPKAEALL 4.000 546. 47 75 RPVPNTSDV 4.000 547. 48 41 LSSPSPTAL 4.000548. 49 454 LLQEGELLL 4.000 549. 50 462 LSPHMQKAL 4.000 550.

TABLE XVI HLA Peptide Scoring Results - 205P1B5 - B7, 10-mers Score(Estimate of Half Time of Disassociation of a Start Subsequence MoleculeContaining Rank Position Residue Listing This Subsequence) Seq. ID# 1362 MPHWVRGALL 80.000 551. 2 318 IPSTSLVIPL 80.000 552. 3 5 CPVFLSFTKL80.000 553. 4 380 MNRPPPPVEL 60.000 554. 5 156 AVTHMTKAHL 60.000 555. 6496 VAMVIDRIFL 54.000 556. 7 190 FPFDQQNCKM 20.000 557. 8 498 MVIDRIFLWL20.000 558. 9 302 SVLLSLTVFL 20.000 559. 10 510 IVCFLGTIGL 20.000 560.11 323 LVIPLIGEYL 20.000 561. 12 257 YAFVIRRLPL 18.000 562. 13 453ALLQEGELLL 12.000 563. 14 445 GASGPKAEAL 12.000 564. 15 32 PPPRAPGDPL12.000 565. 16 369 ALLGCVPRWL 12.000 566. 17 452 EALLQEGELL 12.000 567.18 446 ASGPKAEALL 12.000 568. 19 102 NQMMTTNVWL 12.000 569. 20 365WVRGALLGCV 10.000 570. 21 370 LLGCVPRWLL 9.000 571. 22 384 PPPVELCHPL8.000 572. 23 264 LPLFYTINLI 8.000 573. 24 138 EMIWIPDIVL 6.000 574. 25388 ELCHPLRLKL 6.000 575. 26 361 TMPHWVRGAL 6.000 576. 27 280 SCLTVLVFYL4.000 577. 28 273 IIPCLLISCL 4.000 578. 29 324 VIPLIGEYLL 4.000 579. 30276 CLLISCLTVL 4.000 580. 31 77 VPNTSDVVIV 4.000 581. 32 471 EGVHYIADHL4.000 582. 33 298 TLCISVLLSL 4.000 583. 34 461 LLSPHMQKAL 4.000 584. 35204 WTYDKAKIDL 4.000 585. 36 516 TIGLFLPPFL 4.000 586. 37 296 KITLCISVLL4.000 587. 38 304 LLSLTVFLLL 4.000 588. 39 332 LLFTMIFVTL 4.000 589. 40303 VLLSLTVFLL 4.000 590. 41 269 TINLIIPCLL 4.000 591. 42 268 YTINLIIPCL4.000 592. 43 263 RLPLFYTINL 4.000 593. 44 433 VGTLCSHGHL 4.000 594. 4575 RPVPNTSDVV 4.000 595. 46 11 FTKLSLWWLL 4.000 596. 47 255 VTYAFVIRRL4.000 597. 48 53 GSHTETEDRL 4.000 598. 49 341 LSIVITVFVL 4.000 599. 5039 DPLSSPSPTA 3.000 600.

TABLE XVII HLA Peptide Scoring Results - 205P1B5 - B35, 9-mers Score(Estimate of Half Time of Disassociation Start Subsequence of a MoleculeContaining Rank Position Residue Listing This Subsequence) Seq. ID# 1208 KAKIDLEQM 54.000 601. 2 362 MPHWVRGAL 20.000 602. 3 325 IPLIGEYLL20.000 603. 4 274 IPCLLISCL 20.000 604. 5 264 LPLFYTINL 20.000 605. 6289 LPSDCGEKI 16.000 606. 7 487 SSVKEDWKY 15.000 607. 8 198 KMKFGSWTY12.000 608. 9 131 TSLRVPSEM 10.000 609. 10 110 WLKQEWSDY 9.000 610. 1175 RPVPNTSDV 8.000 611. 12 77 VPNTSDVVI 8.000 612. 13 521 LPPFLAGMI8.000 613. 14 33 PPRAPGDPL 6.000 614. 15 260 VIRRLPLFY 6.000 615. 16 119KLRWNPTDF 6.000 616. 17 305 LSLTVFLLL 5.000 617. 18 181 SSCSIDVTF 5.000618. 19 41 LSSPSPTAL 5.000 619. 20 279 ISCLTVLVF 5.000 620. 21 462LSPHMQKAL 5.000 621. 22 446 ASGPKAEAL 5.000 622. 23 468 KALEGVHYI 4.800623. 24 385 PPVELCHPL 4.000 624. 25 123 NPTDFGNIT 4.000 625. 26 382RPPPPVELC 4.000 626. 27 452 EALLQEGEL 3.000 627. 28 11 FTKLSLWWL 3.000628. 29 217 EQTVDLKDY 3.000 629. 30 496 VAMVIDRIF 3.000 630. 31 9LSFTKLSLW 2.500 631. 32 396 KLSPSYHWL 2.000 632. 33 430 APSVGTLCS 2.000633. 34 372 GCVPRWLLM 2.000 634. 35 520 FLPPFLAGM 2.000 635. 36 280SCLTVLVFY 2.000 636. 37 323 LVIPLIGEY 2.000 637. 38 398 SPSYHWLES 2.000638. 39 214 EQMEQTVDL 2.000 639. 40 237 TGTYNSKKY 2.000 640. 41 296KITLCISVL 2.000 641. 42 2 GPSCPVFLS 2.000 642. 43 174 VPPAIYKSS 2.000643. 44 454 LLQEGELLL 2.000 644. 45 13 KLSLWWLLL 2.000 645. 46 39DPLSSPSPT 2.000 646. 47 232 AIVNATGTY 2.000 647. 48 488 SVKEDWKYV 1.800648. 49 415 VVVEEEDRW 1.500 649. 50 80 TSDVVIVRF 1.500 650.

TABLE XVIII HLA Peptide Scoring Results - 205P1B5 - B35, 10-mers Score(Estimate of Half Time of Disassociation Start Subsequence of a MoleculeContaining Rank Position Residue Listing This Subsequence) Seq. ID# 1190 FPFDQQNCKM 80.000 651. 2 325 IPLIGEYLLF 30.000 652. 3 362 MPHWVRGALL20.000 653. 4 318 IPSTSLVIPL 20.000 654. 5 2 GPSCPVFLSF 20.000 655. 6 5CPVFLSFTKL 20.000 656. 7 486 DSSVKEDWKY 15.000 657. 8 180 KSSCSIDVTF10.000 658. 9 356 SPSTHTMPHW 10.000 659. 10 183 CSIDVTFFPF 10.000 660.11 279 ISCLTVLVFY 10.000 661. 12 466 MQKALEGVHY 9.000 662. 13 75RPVPNTSDVV 8.000 663. 14 264 LPLFYTINLI 8.000 664. 15 181 SSCSIDVTFF7.500 665. 16 77 VPNTSDVVIV 6.000 666. 17 231 WAIVNATGTY 6.000 667. 18341 LSIVITVFVL 5.000 668. 19 301 ISVLLSLTVF 5.000 669. 20 53 GSHTETEDRL5.000 670. 21 446 ASGPKAEALL 5.000 671. 22 452 EALLQEGELL 4.500 672. 23496 VAMVIDRIFL 4.500 673. 24 61 RLFKHLFRGY 4.000 674. 25 327 LIGEYLLFTM4.000 675. 26 36 APGDPLSSPS 4.000 676. 27 123 NPTDFGNITS 4.000 677. 28289 LPSDCGEKIT 4.000 678. 29 445 GASGPKAEAL 3.000 679. 30 394 RLKLSPSYHW3.000 680. 31 380 MNRPPPPVEL 3.000 681. 32 202 GSWTYDKAKI 3.000 682. 33257 YAFVIRRLPL 3.000 683. 34 139 MIWIPDIVLY 3.000 684. 35 11 FTKLSLWWLL3.000 685. 36 9 LSFTKLSLWW 2.500 686. 37 252 YPDVTYAFVI 2.400 687. 38391 HPLRLKLSPS 2.000 688. 39 263 RLPLFYTINL 2.000 689. 40 498 MVIDRIFLWL2.000 690. 41 79 NTSDVVIVRF 2.000 691. 42 174 VPPAIYKSSC 2.000 692. 43398 SPSYHWLESN 2.000 693. 44 204 WTYDKAKIDL 2.000 694. 45 236 ATGTYNSKKY2.000 695. 46 130 ITSLRVPSEM 2.000 696. 47 371 LGCVPRWLLM 2.000 697. 4832 PPPRAPGDPL 2.000 698. 49 45 SPTALPQGGS 2.000 699. 50 259 FVIRRLPLFY2.000 700.

TABLE XIX Motifs and Post-translational Modifications of 205P1B5N-glycosylation site Number of matches: 3 1  79-82 NTSD (SEQ ID NO.:720) 2 129-132 NITS (SEQ ID NO.: 721) 3 235-238 NATG (SEQ ID NO.: 722)Protein kinase C phosphorylation site Number of matches: 3 1 132-134 SLR2 242-244 SKK 3 488-490 SVK Casein kinase II phosphorylation site Numberof matches: 4 1  54-57 SHTE (SEQ ID NO.: 723) 2  56-59 TETE (SEQ ID NO.:724) 3 406-409 SNVD (SEQ ID NO.: 725) 4 488-491 SVKE (SEQ ID NO.: 726)Tyrosine kinase phosphorylation site 468-475 KALEGVHY (SEQ ID NO.: 727)N-myristoylation site Number of matches: 7 1  25-30 GGEEAK (SEQ ID NO.:728) 2  52-57 GGSHTE (SEQ ID NO.: 729) 3  89-94 GLSIAQ (SEQ ID NO.: 730)4 128-133 GNITSL (SEQ ID NO.: 731) 5 238-243 GTYNSK (SEQ ID NO.: 732) 6368-373 GALLGC (SEQ ID NO.: 733) 7 434-439 GTLCSH (SEQ ID NO.: 734)Neurotransmitter-gated ion-channels signature 183-197 CSIDVTFFPFDQQNC(SEQ ID NO.: 735) BLOCKS Neurotransmitter-gated ion-channel between aa168-206 Neurotransmitter-gated ion-channel between aa 252-296 PRINTSNicotinic acetylcholine receptor signature between aa 163-181- Nicotinicacetylcholine receptor signature between aa 93-109 PfamNeurotransmitter-gated ion-channel ligand binding domain between aa59-265 Neurotransmitter-gated ion-channel transmembrane region betweenaa 272-520

TABLE XX Frequently Occurring Motifs avrg. % Name identity DescriptionPotential Function zf-C2H2 34% Zinc finger, C2H2 type Nucleicacid-binding protein functions as transcription factor, nuclear locationprobable cytochrome_b_N 68% Cytochrome b(N- membrane bound oxidase,generate terminal)/b6/petB superoxide ig 19% Immunoglobulin domaindomains are one hundred amino acids long and include a conservedintradomain disulfide bond. WD40 18% WD domain, G-beta repeat tandemrepeats of about 40 residues, each containing a Trp-Asp motif. Functionin signal transduction and protein interaction PDZ 23% PDZ domain mayfunction in targeting signaling molecules to sub-membranous sites LRR28% Leucine Rich Repeat short sequence motifs involved in protein-protein interactions pkinase 23% Protein kinase domain conservedcatalytic core common to both serine/threonine and tyrosine proteinkinases containing an ATP binding site and a catalytic site PH 16% PHdomain pleckstrin homology involved in intracellular signaling or asconstituents of the cytoskeleton EGF 34% EGF-like domain 30-40amino-acid long found in the extracellular domain of membrane-boundproteins or in secreted proteins rvt 49% Reverse transcriptase(RNA-dependent DNA polymerase) ank 25% Ank repeat Cytoplasmic protein,associates integral membrane proteins to the cytoskeleton oxidored_q132% NADH- Ubiquinone/plastoquinone membrane associated. Involved inproton (complex I), various chains translocation across the membraneefhand 24% EF hand calcium-binding domain, consists of a12 residue loopflanked on both sides by a 12 residue alpha-helical domain rvp 79%Retroviral aspartyl protease Aspartyl or acid proteases, centered on acatalytic aspartyl residue Collagen 42% Collagen triple helix repeatextracellular structural proteins involved in (20 copies) formation ofconnective tissue. The sequence consists of the G-X-Y and thepolypeptide chains forms a triple helix. fn3 20% Fibronectin type IIIdomain Located in the extracellular ligand-binding region of receptorsand is about 200 amino acid residues long with two pairs of cysteinesinvolved in disulfide bonds 7tm_1 19% 7 transmembrane receptor sevenhydrophobic transmembrane regions, (rhodopsin family) with theN-terminus located extracellularly while the C-terminus is cytoplasmic.Signal through G proteins

TABLE XXI Properties of 205P1B5 Bioinformatic Feature Program World wideweb URL Outcome ORF (includes stop codon) ORF finder ncbi.nlm.nih.gov/See FIG. 2; 555-2144 # of amino acids 529 amino acids Transmembraneregion TM Pred ch.embnet.org/ 5 transmembrane domains HMMTopenzim.hu/hmmtop/ 4 transmembrane domains Sosui genome.ad.jp/SOSui/ 5transmembrane domains TMHMM cbs.dtu.dk/services/TMHMM 5 transmembranedomains Signal Peptide Signal P cbs.dtu.dk/services/SignalP/ Signalsequence aa 1-26 pI pI/MW tool expasy.ch/tools/ pI 5.69 Molecular weightpI/MW tool expasy.ch/tools/ 59.76 kDa Localization PSORTpsort.nibb.ac.jp/ plasma membrane 0.600 mitochondrial membrane 0.400PSORT II psort.nibb.ac.jp/ Plasma membrane 22.2% endoplasmic reticulum33.3% Motifs Pfam sanger.ac.uk/Pfam/ Neurotransmitter-gated ion- channelPrints biochem.ucl.ac.uk/ Nicotinic acetylcholine receptor signatureBlocks blocks.fhcrc.org/ Neurotransmitter-gated ion- channel Prositegenome.ad.jp/ Neurotransmitter-gated ion- channels

1. An isolated polynucleotide that encodes a protein comprising SEQ IDNO:
 702. 2. The polynucleotide of claim 1, wherein the polynucleotidecomprises SEQ ID NO:701 from residue 555 to
 2144. 3. A pharmaceuticalcomposition comprising the polynucleotide of claim 1 and apharmaceutically acceptable carrier.
 4. The polynucleotide of claim 1,wherein the polynucleotide further comprises an expression vector. 5.The polynucleotide of claim 4, wherein the expression vector is a viralvector.
 6. The polynucleotide of claim 5, wherein the viral expressionvector is selected from the group consisting of vaccinia virus, fowlpoxvirus, canarypox virus, adenovirus, influenza virus, poliovirus,adeno-associated virus, lentivirus, and sindbis virus.
 7. An isolatedhost cell that contains an expression vector of claim
 4. 8. A processfor producing a 205P1B5 protein comprising culturing a host cell ofclaim 7 under conditions sufficient for the production of the protein,and recovering the protein comprising SEQ ID NO: 702 from the culture.